Flow cells

ABSTRACT

An example of a flow cell includes a substrate; a first primer set attached to a first region on the substrate, the first primer set including an un-cleavable first primer and a cleavable second primer; and a second primer set attached to a second region on the substrate, the second primer set including a cleavable first primer and an un-cleavable second primer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/692,511, filed Jun. 29, 2018 and of U.S. Provisional ApplicationSer. No. 62/743,373, filed Oct. 9, 2018; the content of each of which isincorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted herewith via EFS-Web is herebyincorporated by reference in its entirety. The name of the file isILI172APCT_IP-1806-PCT_Sequence_Listing_ST25.txt, the size of the fileis 551 bytes, and the date of creation of the file is May 20, 2019.

BACKGROUND

Some available platforms for sequencing nucleic acids utilize asequencing-by-synthesis approach. With this approach, a nascent strandis synthesized, and the addition of each monomer (e.g., nucleotide) tothe growing strand is detected optically and/or electronically. Becausea template strand directs synthesis of the nascent strand, one can inferthe sequence of the template DNA from the series of nucleotide monomersthat were added to the growing strand during the synthesis. In someexamples, paired-end sequencing may be used, where forward strands aresequenced and removed, and then reverse strands are constructed andsequenced.

INTRODUCTION

A first aspect disclosed herein is flow cell, comprising: a substrate; afirst primer set attached to a first region on the substrate, the firstprimer set including an un-cleavable first primer and a cleavable secondprimer; and a second primer set attached to a second region on thesubstrate, the second primer set including a cleavable first primer andan un-cleavable second primer.

In an example of the first aspect, the first region includes a materialhaving a first functional group; and the second region includes amaterial having a second functional group that is different than thefirst functional group.

In an example of the first aspect, the flow cell further comprises a gapseparating the first primer set from the second primer set.

In an example of the first aspect, the substrate includes depressionsseparated by interstitial regions; and each of the depressions includes:the first region located at a first portion; and the second regionlocated at a second portion. In one version of this example, the flowcell may further comprise a gap separating the first region from thesecond region. In another version of this example, the first region andthe second region partially overlap. In still another version of thisexample, the first and second portions have different depths. In afurther version of this example, the first and second regions aredifferent blocks of a block co-polymer.

In an example of the first aspect, the substrate includes depressionsseparated by interstitial regions; each of the depressions includes thefirst region; and the second region is located on at least some of theinterstitial regions.

In an example of the first aspect, the first region includes a firstpolymer and the first primer set is grafted to the first polymer; andthe second region includes a second polymer and the second primer set isgrafted to the second polymer. In one version of this example, the flowcell further comprises a protective coating on the first primer set andon the first polymer. In another version of this example, the firstpolymer is a first layer on the substrate; the second polymer is asecond layer on the first layer; the flow cell further comprises: apassivation resin on the second layer; and features defined in thepassivation resin, the second polymer and the first polymer; and each ofthe first and second primer sets is exposed at each of the features.

In an example of the first aspect, the substrate includes depressionsseparated by interstitial regions; each of the depressions includes: afirst portion where the first region is located; and a second portion;and the flow cell further comprises a bead located in the secondportion, wherein the second region is at a surface of the bead.

In an example of the first aspect, the cleavable first primer includes afirst cleavage site, the cleavable second primer includes a secondcleavage site, and the first and second cleavage sites are of anidentical type. In one version of this example, each of the un-cleavablefirst primer, the cleavable second primer, the cleavable first primer,and the cleavable second primer includes a respective linker; the firstcleavage site of the first cleavable primer is located along its linker;and the second cleavage site of the second cleavable primer is locatedalong its linker.

In an example of the first aspect, the cleavable first primer includes afirst cleavage site, the cleavable second primer includes a secondcleavage site, and the first and second cleavage sites are of adifferent type. In one version of this example, each of the un-cleavablefirst primer, the cleavable second primer, the cleavable first primer,and the cleavable second primer includes a respective linker; the firstcleavage site of the first cleavable primer is located along its linker;and the second cleavage site of the second cleavable primer is locatedalong its linker.

In an example of the first aspect, the first primer set is attached to afirst support structure; the first region is a first capture site thatis attached to the first support structure; the second primer set isattached to a second support structure that is different than the firstsupport structure; and the second region is a second capture site thatis attached to the second support structure.

It is to be understood that any features of the first aspect disclosedherein may be combined together in any desirable manner and/orconfiguration.

A second aspect disclosed herein is a flow cell comprising a firstsubstrate; a first primer set attached to the first substrate, the firstprimer set including an un-cleavable first primer and a cleavable secondprimer; a second substrate opposed to the first substrate; and a secondprimer set attached to the second substrate, the second primer setincluding a cleavable first primer and an un-cleavable second primer.

It is to be understood that any features of the second aspect may becombined together in any desirable manner. Moreover, it is to beunderstood that any combination of features of the first aspect and/orof the second aspect may be used together, and/or may be combined withany of the examples disclosed herein.

A third aspect disclosed herein is a kit comprising a flow cellincluding: a substrate including depressions separated by interstitialregions; a first polymer layer in each of the depressions, wherein somefunctional groups of the first polymer layer are capped; a first primerset attached to other functional groups of first polymer layer in eachof the depressions; and a second polymer layer on the interstitialregions; and a priming fluid including: a fluid carrier; and a secondprimer set that is different from the first primer set.

In an example of the third aspect, the first primer set includes anun-cleavable first primer and a cleavable second primer; and the secondprimer set includes a cleavable first primer and an un-cleavable secondprimer.

It is to be understood that any features of the third aspect may becombined together in any desirable manner. Moreover, it is to beunderstood that any combination of features of the third aspect and/orof the second aspect and/or of the first aspect may be used together,and/or may be combined with any of the examples disclosed herein.

A fourth aspect disclosed herein is a method comprising introducing atemplate fluid to a flow cell including: a substrate includingdepressions separated by interstitial regions; a first polymer layer ineach of the depressions, wherein exposed functional groups of the firstpolymer layer are capped; a first primer set attached to the firstpolymer layer in each of the depressions, the first primer set includinga cleavable first primer and an un-cleavable second primer; and a secondpolymer layer on the interstitial regions; whereby a template from thetemplate fluid is amplified to form a cluster in at least some of thedepressions; introducing a priming fluid, including an un-cleavablefirst primer and a cleavable second primer, to the flow cell, wherebythe un-cleavable first primer and the cleavable second primer graft tothe second polymer layer; and initiating bridge amplification from thecluster to the un-cleavable first primer and a cleavable second primer,thereby forming a second cluster on at least some of the interstitialregions.

It is to be understood that any features of the fourth aspect may becombined together in any desirable manner. Moreover, it is to beunderstood that any combination of features of the fourth aspect and/orof the third aspect and/or of the second aspect and/or of the firstaspect may be used together, and/or may be combined with any of theexamples disclosed herein.

A fifth aspect disclosed herein is a method, comprising: introducing atemplate fluid to a flow cell including: a substrate includingdepressions separated by interstitial regions; a first polymer layer ineach of the depressions; a first primer set attached to the firstpolymer layer, the first primer set including a cleavable first primerand an un-cleavable second primer; an optional protective coating layeron the first polymer layer and on the first primer set; a second polymerlayer on the interstitial regions; and a second primer set attached tothe second polymer layer, the second primer set including anun-cleavable first primer and a cleavable second primer; whereby atemplate from the template fluid is amplified to form a cluster in atleast some of the depressions and on at least some of the interstitialregions; and cleaving the cleavable first primer and the cleavablesecond primer.

It is to be understood that any features of the fifth aspect may becombined together in any desirable manner. Moreover, it is to beunderstood that any combination of features of the fifth aspect and/orof the third aspect and/or of the second aspect and/or of the firstaspect may be used together, and/or may be combined with any of theexamples disclosed herein.

A sixth aspect disclosed herein is a flow cell comprising a support; apatterned resin on the support, the patterned resin including firstdepressions and second depressions separated by interstitial regions,the first depressions having smaller opening dimensions than the seconddepressions; a first primer set attached in at least some of the firstdepressions; and a functionalized bead respectively positioned in atleast some of the second depressions, the functionalized bead includinga second primer set attached at a surface of a core structure, whereinthe second primer set is different than the first primer set.

In an example of the sixth aspect, the core structure of thefunctionalized bead is selected from the group consisting of silicondioxide, a superparamagnetic material, polystyrene, and an acrylate.

In an example of the sixth aspect, the patterned resin is selected fromthe group consisting of a polyhedral oligomeric silsesquioxane resin(POSS)-based resin, an epoxy resin, a poly(ethylene glycol) resin, apolyether resin, an acrylic resin, an acrylate resin, a methacrylateresin, and combinations thereof.

In an example of the sixth aspect, the flow cell further comprises apolymer in the first depressions and in the second depressions, andwherein the first primer set is attached to the polymer in the at leastsome of the first depressions. In one version of this example, thefunctionalized bead is positioned on the polymer in the at least some ofthe second depressions. In another version of this example, the firstprimer set is also attached to the polymer in the at least some of thesecond depressions; and the functionalized bead is positioned on thefirst primer set in the at least some of the second depressions.

In an example of the sixth aspect, the first primer set includes a firstprimer and a uracil-modified second primer; and the second primer setincludes a uracil-modified first primer and a second primer.

It is to be understood that any features of the sixth aspect disclosedherein may be combined together in any desirable manner and/orconfiguration.

A seventh aspect disclosed herein is a flow cell comprising a support; apatterned resin on the support, the patterned resin includingdepressions separated by interstitial regions; a first primer setattached to at least some of the depressions; and a functionalized beadpositioned in the at least some of the depressions so that at least someprimers of the first primer set are exposed, the functionalized beadincluding a second primer set attached at a surface of a core structure,wherein the second primer set is different than the first primer set.

In an example of the seventh aspect, each of the depressions includes afirst portion with a first opening dimension that is larger than orequal to a diameter of the functionalized bead, and a second portionwith a second opening dimension that is smaller than the diameter of thefunctionalized bead; and the functionalized bead is positioned in thefirst portion of each of the at least some of the depressions.

In an example of the seventh aspect, the core structure of thefunctionalized bead is selected from the group consisting of silicondioxide, a superparamagnetic material, polystyrene, and an acrylate.

In an example of the seventh aspect, the patterned resin is selectedfrom the group consisting of a polyhedral oligomeric silsesquioxaneresin (POSS)-based resin, an epoxy resin, a poly(ethylene glycol) resin,a polyether resin, an acrylic resin, an acrylate resin, a methacrylateresin, and combinations thereof.

In an example of the seventh aspect, the flow cell further comprises apolymer in the depressions. In one version of this aspect, the firstprimer set is attached to a portion of the polymer unoccupied by thefunctionalized bead. In one example of this version, the first primerset is attached to the polymer in the depressions; and thefunctionalized bead is positioned on some other primers of the firstprimer set.

It is to be understood that any features of the seventh aspect disclosedherein may be combined together in any desirable manner and/orconfiguration.

An eighth aspect disclosed herein is a method comprising selectivelyapplying a polymer in depressions of a patterned resin on a support;grafting a first primer set to the polymer in at least some of thedepressions; and before or after grafting the first primer set,depositing functionalized beads i) in a portion of each of the at leastsome of the depressions, or ii) in second depressions having largeropening dimensions than the at least some of the depressions, thefunctionalized beads including a second primer set attached at a surfaceof a core structure, wherein the first and second primer sets aredifferent.

In an example of the eighth aspect, wherein prior to depositing thefunctionalized beads, the method further comprises forming thefunctionalized beads by attaching the second primer set to the corestructure.

In an example of the eighth aspect, the portion of each of the at leastsome of the depressions has an opening dimension that is larger than orequal to a diameter of each of the functionalized beads; the at leastsome of the depressions include a second portion interconnected with theportion, where the second portion has a second opening dimension that issmaller than the diameter of each of the functionalized beads; and thefunctionalized beads self-assemble into the portion of each of the atleast some of the depressions by size exclusion.

In an example of the eighth aspect, wherein prior to selectivelyapplying the polymer, the method further comprises forming the patternedresin on the support by: depositing a resin on the support; andpatterning the resin using nanoimprint lithography.

It is to be understood that any features of the eighth aspect may becombined together in any desirable manner. Moreover, it is to beunderstood that any combination of features of the eighth aspect and/orof the seventh aspect and/or of the sixth aspect and/or of the firstaspect may be used together, and/or may be combined with any of theexamples disclosed herein.

A ninth aspect disclosed herein is a flow cell comprising a support; apatterned resin on the support, the patterned resin includingdepressions separated by interstitial regions; a block copolymer on thepatterned resin in the depressions, each block of the block copolymerhaving a block-specific functional group that is different from theblock-specific functional group of each other block of the blockcopolymer; and a primer attached to the block-specific functional groupof at least one of the blocks.

In an example of the ninth aspect, the patterned resin is selected fromthe group consisting of a polyhedral oligomeric silsesquioxane resin(POSS)-based resin, an epoxy resin, a poly(ethylene glycol) resin, apolyether resin, an acrylic resin, an acrylate resin, a methacrylateresin, and combinations thereof. In one version of this example, thepatterned resin is the POSS-based resin, and wherein the POSS-basedresin is a cross-linked epoxy POSS resin. In another version of thisexample, the block copolymer includes: a first block including anacrylamide monomer having an amino group as its block-specificfunctional group; and a second block including an azido acetamido pentylacrylamide monomer having an azido group as its block-specificfunctional group. In an example of this other version, the blockcopolymer is:

wherein R is hydrogen or a polymer initiating species end group, nranges from 1 to 10,000, and m ranges from 1 to 10,000. In still anotherversion of this example, the block copolymer is:

wherein n ranges from 1 to 10,000, and m ranges from 1 to 10,000.

In an example of the ninth aspect, the patterned resin is an amorphousfluoropolymer. In one version of this example, the block copolymerincludes: a first block including a monomer having a trifluoromethylgroup as its block-specific functional group; and a second blockincluding a monomer having a primer-grafting functional group as itsblock-specific functional group.

In an example of the ninth aspect, the block copolymer includes: a firstblock including a monomer having a primer-grafting functional group asits block-specific functional group; and a second block including amonomer to adjust an interaction parameter to drive phase separation ofthe first and second blocks. In one version of this example, theprimer-grafting functional group is an azido group; and theblock-specific functional group of the monomer of the second block isselected from the group consisting of an amino group, an alcohol group,an aryl group, and a charged group.

In an example of the ninth aspect, the block copolymer is a terpolymerincluding a first block, a second block, and a third block; theblock-specific functional group of the first block is attached to thepatterned resin; the block-specific functional group of the second blockis attached to the primer; and the block-specific functional group ofthe third block is attached to an other primer that is different thanthe primer, or to an enzyme.

In an example of the ninth aspect, the block copolymer is a terpolymerincluding a first block, a second block, and a third block; theblock-specific functional group of the first block is attached to thepatterned resin; the block-specific functional group of the second blockis attached to the primer; and the block-specific functional group ofthe third block affects a surface free energy of the block copolymer oraffects stability of the block copolymer.

In an example of the ninth aspect, wherein the depressions are selectedfrom the group consisting of wells and trenches.

In an example of the ninth aspect, the patterned resin and the blockcopolymer each have a surface free energy within a range of from about25 mN/m to about 50 mN/m.

It is to be understood that any features of the ninth aspect disclosedherein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the ninth aspect and/or of the first aspect may be usedtogether, and/or may be combined with any of the examples disclosedherein.

A tenth aspect disclosed herein is a flow cell comprising a support; apatterned polyhedral oligomeric silsesquioxane (POSS)-based resin on thesupport, the patterned POSS-based resin including depressions separatedby interstitial regions; segregated block copolymer on the patternedPOSS-based resin in the depressions, wherein one block of the segregatedblock copolymer has a functional group attached to the patternedPOSS-based resin and an other block of the segregated block copolymerhas an other functional group; and a primer attached to the otherfunctional group.

In an example of the tenth aspect, the segregated block copolymer isselected from the group consisting of:

wherein n ranges from 1 to 10,000, and m ranges from 1 to 10,000; and

wherein R is hydrogen or a polymer initiating species end group, nranges from 1 to 10,000, and m ranges from 1 to 10,000.

It is to be understood that any features of the tenth aspect disclosedherein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the tenth aspect and/or of the ninth aspect and/or of thefirst aspect may be used together, and/or may be combined with any ofthe examples disclosed herein.

An eleventh aspect disclosed herein is a method comprising patterning aresin to form a patterned resin including depressions separated byinterstitial regions; introducing a solution including a block copolymeron the patterned resin, each block of the block copolymer having ablock-specific functional group that is different from theblock-specific functional group of each other block of the blockcopolymer; exposing the solution to solvent vapor annealing, whereby theblock copolymer phase separates and self-assembles in the depressions;and grafting a primer to the block-specific functional group of at leastone of the blocks.

In an example of the eleventh aspect, patterning the resin involvesnano-imprint lithography.

In an example of the eleventh aspect, the solution including the blockcopolymer has a Flory-Huggins interaction parameter ranging from about0.04 to about 0.30.

In an example of the eleventh aspect, wherein prior to grafting, themethod further comprises exposing the patterned resin, including thephase separated and self-assembled block copolymer in the depressions,to a curing process.

It is to be understood that any features of the eleventh aspectdisclosed herein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the eleventh aspect and/or of the tenth aspect and/or of theninth aspect and/or of the first aspect may be used together, and/or maybe combined with any of the examples disclosed herein.

A twelfth aspect disclosed herein is a method comprising applying afirst functionalized layer on a substrate; patterning the firstfunctionalized layer, thereby forming a first functionalized regioncovered by a photoresist; applying a second functionalized layer on thephotoresist and portions of the substrate; lifting off the photoresistand any of the second functionalized layer thereon; removing a portionof the second functionalized layer, thereby forming a secondfunctionalized region adjacent to the first functionalized region; andattaching a first primer set to the first functionalized layer or thefirst functionalized region and a second primer set to the secondfunctionalized layer or the second functionalized region, wherein thefirst primer set is different from the second primer set.

In an example of the twelfth aspect, the attaching of the first primerset involves pre-grafting an un-cleavable first primer and a cleavablesecond primer to the first functionalized layer before the firstfunctionalized layer is applied; and the attaching of the second primerset involves pre-grafting a cleavable first primer and an un-cleavablesecond primer to the second functionalized layer before the secondfunctionalized layer is applied.

In an example of the twelfth aspect, the attaching of the first primerset involves grafting an un-cleavable first primer and a cleavablesecond primer to the first functionalized layer after its application;and the attaching of the second primer set involves grafting a cleavablefirst primer and an un-cleavable second primer to the secondfunctionalized layer after its application.

In an example of the twelfth aspect, the method further comprises:depositing, respectively, a first self-assembled monolayer on the firstfunctionalized region and a second self-assembled monolayer on thesecond functionalized region; wherein the attaching of the first primerset includes grafting an un-cleavable first primer and a cleavablesecond primer to the first self-assembled monolayer; and wherein theattaching of the second primer set includes grafting a cleavable firstprimer and an un-cleavable second primer to the second self-assembledmonolayer.

In an example of the twelfth aspect, wherein the removing involves:applying a second photoresist on the first functionalized region and asecond portion of the second functionalized layer that is to become thesecond functionalized region; and etching the portion of the secondfunctionalized layer.

In an example of the twelfth aspect, the substrate includes a resin on asupport; the resin includes depressions separated by interstitialregions; the first functionalized region is on a first portion of eachdepression; the second functionalized layer is on a second portion ofeach depression and on the interstitial regions; and the removinginvolves polishing the second functionalized layer from the interstitialregions.

In an example of the twelfth aspect, the substrate includes a resin on asupport; the resin includes multi-level depressions separated byinterstitial regions; the first functionalized region is at a firstlevel of each multi-level depression; and prior to applying the secondfunctionalized layer, the method further comprises: applying asacrificial layer on the photoresist and portions of the resin; removingthe sacrificial layer from the portions of the resin; and removing aregion of the resin from the multi-layer depression to create an areathat is adjacent to the first functionalized region; and the secondfunctionalized layer is applied on the sacrificial layer on thephotoresist, on the area, and on the interstitial regions.

It is to be understood that any features of the twelfth aspect disclosedherein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the twelfth aspect and/or of the first aspect and/or of thesecond aspect may be combined with any of the examples disclosed herein.

A thirteenth aspect disclosed herein is a method comprising applying afirst photoresist on a substrate so that a first substrate portion isexposed; applying a first functionalized layer on the photoresist andthe first substrate portion; lifting off the photoresist and any of thefirst functionalized layer thereon, thereby forming a firstfunctionalized region on the first substrate portion; applying a secondphotoresist on the first functionalized region and on the substrate sothat a second substrate portion adjacent to the first functionalizedregion is exposed; applying a second functionalized layer on the secondphotoresist and the second substrate portion; lifting off the secondphotoresist and any of the second functionalized layer thereon, therebyforming a second functionalized region adjacent to the firstfunctionalized region; and attaching a first primer set to the firstfunctionalized layer or the first functionalized region and a secondprimer set to the second functionalized layer or the secondfunctionalized region, wherein the first primer set is different fromthe second primer set.

In an example of the thirteenth aspect, the attaching of the firstprimer set involves pre-grafting an un-cleavable first primer and acleavable second primer to the first functionalized layer before thefirst functionalized layer is applied; and the attaching of the secondprimer set involves pre-grafting a cleavable first primer and anun-cleavable second primer to the second functionalized layer before thesecond functionalized layer is applied.

In an example of the thirteenth aspect, the attaching of the firstprimer set involves grafting an un-cleavable first primer and acleavable second primer to the first functionalized layer after itsapplication; and the attaching of the second primer set involvesgrafting a cleavable first primer and an un-cleavable second primer tothe second functionalized layer after its application.

In an example of the thirteenth aspect, the method further comprisesdepositing, respectively, a first self-assembled monolayer on the firstfunctionalized region and a second self-assembled monolayer on thesecond functionalized region; wherein the attaching of the first primerset includes grafting an un-cleavable first primer and a cleavablesecond primer to the first self-assembled monolayer; and wherein theattaching of the second primer set includes grafting a cleavable firstprimer and an un-cleavable second primer to the second self-assembledmonolayer.

It is to be understood that any features of the thirteenth aspectdisclosed herein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the thirteenth aspect and/or of the first aspect and/or ofthe second aspect may be combined with any of the examples disclosedherein.

A fourteenth aspect disclosed herein is a method comprising applying afirst functionalized layer on a substrate including trenches separatedby interstitial regions and a sacrificial material region in a firstportion of each of the trenches; patterning the first functionalizedlayer, thereby forming a first functionalized region covered by aphotoresist in a second portion of each of the trenches; removing thesacrificial material region to expose the first portion of each of thetrenches; applying a second functionalized layer on the interstitialregions, on the first portion, and on the photoresist; lifting off thephotoresist and any of the second functionalized layer thereon; removingany of the second functionalized layer from the interstitial regions,whereby a second functionalized region remains in the first portion ofeach of the trenches; applying a second photoresist in a pattern ofspatially separated stripes that are at least substantiallyperpendicular to the trenches; removing portions of the firstfunctionalized regions and the second functionalized regions that areexposed between the spatially separated stripes; removing the secondphotoresist; and attaching a first primer set to the firstfunctionalized layer or the first functionalized regions and a secondprimer set to the second functionalized layer or the secondfunctionalized regions, wherein the first primer set is different fromthe second primer set.

In an example of the fourteenth aspect, the attaching of the firstprimer set involves pre-grafting an un-cleavable first primer and acleavable second primer to the first functionalized layer before thefirst functionalized layer is applied; and the attaching of the secondprimer set involves pre-grafting a cleavable first primer and anun-cleavable second primer to the second functionalized layer before thesecond functionalized layer is applied.

In an example of the fourteenth aspect, the attaching of the firstprimer set involves grafting an un-cleavable first primer and acleavable second primer to the first functionalized layer after itsapplication; and the attaching of the second primer set involvesgrafting a cleavable first primer and an un-cleavable second primer tothe second functionalized layer after its application.

In an example of the fourteenth aspect, the substrate includes a secondsacrificial material region in the second portion of each of thetrenches; the substrate, the sacrificial material region, and the secondsacrificial material region have different etch rates; and prior toapplying the first functionalized layer, the method further comprisesremoving the second sacrificial material region from the second portionof each of the trenches. In one version of this example, wherein priorto removing the second sacrificial material region, the method furthercomprises forming the sacrificial material region and second sacrificialmaterial region by: applying a sacrificial material on the substrateincluding the trenches separated by the interstitial regions; removing aportion of the sacrificial material such that a region of thesacrificial material remains directly adjacent to each sidewall of eachof the trenches; applying a second sacrificial material on the substrateand on the sacrificial material regions; removing a portion of thesecond sacrificial material such that a region of the second sacrificialmaterial remains directly adjacent to each of the sacrificial materialregions; and applying a material to fill any spaces between the secondsacrificial material regions. In one example of this version, thesubstrate is a multi-layer substrate; the trenches are defined in anoutermost layer of the multi-layer substrate; and the material and theoutermost layer are the same.

It is to be understood that any features of the fourteenth aspectdisclosed herein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the fourteenth aspect and/or of the first aspect and/or ofthe second aspect may be combined with any of the examples disclosedherein.

A fifteenth aspect disclosed herein is a method comprising applying asacrificial material to a substrate including depressions separated byfirst interstitial regions, wherein each depression includes a deepportion and a shallow portion defined by a step portion, and wherein thesacrificial layer partially fills the deep portion; sequentiallyremoving a portion of the sacrificial layer and a portion of thesubstrate to form second interstitial regions that are at leastsubstantially level with a remaining portion of the sacrificial layerand to remove the step portion to form an area next to the remainingportion of the sacrificial layer; applying a first functionalized layeron the second interstitial regions, the remaining portion of thesacrificial layer, and the area; applying a photoresist on firstfunctionalized layer; removing a portion of the photoresist and anunderlying portion of the first functionalized layer so that theremaining portion of the sacrificial layer and the second interstitialregions are exposed, and a portion of the first functionalized layerhaving a second portion of the photoresist thereon remain at the area;removing the remaining portion of the sacrificial layer to from a secondarea next to the portion of the first functionalized region; applying asecond functionalized layer to the area, thereby forming a secondfunctionalized region; lifting off the second portion of thephotoresist, thereby forming a first functionalized region; andattaching a first primer set to the first functionalized layer or thefirst functionalized region and a second primer set to the secondfunctionalized layer or the second functionalized region, wherein thefirst primer set is different from the second primer set.

In an example of the fifteenth aspect, the second functionalized layeris also applied to the second portion of the photoresist and the secondinterstitial regions; a first portion of the second functionalized layeris removed with the second portion of the photoresist; and the methodfurther comprising polishing the second functionalized layer from thesecond interstitial regions.

In an example of the fifteenth aspect, the attaching of the first primerset involves pre-grafting an un-cleavable first primer and a cleavablesecond primer to the first functionalized layer before the firstfunctionalized layer is applied; and the attaching of the second primerset involves pre-grafting a cleavable first primer and an un-cleavablesecond primer to the second functionalized layer before the secondfunctionalized layer is applied.

In an example of the fifteenth aspect, the attaching of the first primerset involves grafting an un-cleavable first primer and a cleavablesecond primer to the first functionalized layer after its application;and the attaching of the second primer set involves grafting a cleavablefirst primer and an un-cleavable second primer to the secondfunctionalized layer after its application.

It is to be understood that any features of the fifteenth aspectdisclosed herein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the fifteenth aspect and/or of the first aspect and/or ofthe second aspect may be combined with any of the examples disclosedherein.

A sixteenth aspect disclosed herein is a method comprising imprinting amulti-layer substrate including: a support; a first functionalized layeron the support; a second functionalized layer on the firstfunctionalized layer; and a passivation layer on the secondfunctionalized layer; thereby forming features separated by interstitialregions of the passivation layer, wherein a region of each the first andsecond functionalized layers is exposed at each feature; attaching afirst primer set to the first functionalized layer or the firstfunctionalized region and a second primer set to the secondfunctionalized layer or the second functionalized region, wherein thefirst primer set is different from the second primer set.

In an example of the sixteenth aspect, the attaching of the first primerset involves pre-grafting an un-cleavable first primer and a cleavablesecond primer to the first functionalized layer before the firstfunctionalized layer is incorporated into the multi-layer substrate; andthe attaching of the second primer set involves pre-grafting a cleavablefirst primer and an un-cleavable second primer to the secondfunctionalized layer before the second functionalized layer isincorporated into the multi-layer substrate

In an example of the sixteenth aspect, the attaching of the first primerset involves grafting an un-cleavable first primer and a cleavablesecond primer to the first functionalized region in each depression; andthe attaching of the second primer set involves grafting a cleavablefirst primer and an un-cleavable second primer to the secondfunctionalized region in each depression.

It is to be understood that any features of the sixteenth aspectdisclosed herein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the sixteenth aspect and/or of the first aspect and/or ofthe second aspect may be combined with any of the examples disclosedherein.

A seventeenth aspect disclosed herein is a method comprising imprintinga first resin to form a depression including a deep portion and ashallow portion defined by a step portion, wherein the first resin ispositioned on a sacrificial layer that is positioned on a second resin;etching a first portion of the first resin and a portion of thesacrificial layer underlying the deep portion, thereby exposing aportion of the second resin; etching the step portion, thereby exposinga second portion of the sacrificial layer; applying a firstfunctionalized layer to the portion of the second resin to form a firstfunctionalized region; removing the second portion of the sacrificiallayer, thereby exposing a second portion of the second resin; applying asecond functionalized layer to the second portion of the second resin toform a second functionalized region; and attaching a first primer set tothe first functionalized layer or the first functionalized region and asecond primer set to the second functionalized layer or the secondfunctionalized region, wherein the first primer set is different fromthe second primer set

In an example of the seventeenth aspect, during the applying of thesecond functionalized layer, the second functionalized layer isdeposited on interstitial regions surrounding the depression and is notdeposited on the first functionalized region; and the method furthercomprises polishing the second functionalized layer from theinterstitial regions.

It is to be understood that any features of the seventeenth aspectdisclosed herein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the seventeenth aspect and/or of the first aspect and/or ofthe second aspect may be combined with any of the examples disclosedherein.

An eighteenth aspect disclosed herein is a method comprising attaching afirst primer set to a first support structure; attaching a second primerset to a second support structure, wherein the second primer set and thesecond support structure are different than the first primer set and thefirst support structure; and loading the first and second supportstructures on a substrate surface having a plurality of first capturesites to selectively attach to the first support structures and aplurality of second capture sites to selective attach to the secondsupport structures.

It is to be understood that any features of the eighteenth aspectdisclosed herein may be combined together in any desirable manner and/orconfiguration. Moreover, it is to be understood that any combination offeatures of the eighteenth aspect and/or of the first aspect and/or ofthe second aspect may be combined with any of the examples disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

Features of examples of the present disclosure will become apparent byreference to the following detailed description and drawings, in whichlike reference numerals correspond to similar, though perhaps notidentical, components. For the sake of brevity, reference numerals orfeatures having a previously described function may or may not bedescribed in connection with other drawings in which they appear.

FIGS. 1A through 1D are schematic views of different examples of firstand second primer sets attached to first and second region on asubstrate;

FIG. 2 is a schematic, cross-sectional view of an example of the firstand second regions on a substrate surface;

FIGS. 3A and 3B are, respectively, a schematic, cross-sectional view anda top view of an example of the first and second regions in adepression;

FIGS. 4A through 4C are schematic, cross-sectional views of differentexamples of the first and second regions in the depression;

FIG. 5 is a schematic, cross-sectional view of an example of the firstregion in the depression and the second region on a substrate surface;

FIGS. 6A and 6B are, respectively, a schematic, cross-sectional view anda top view of an example of the first region in the depression and thesecond region on a substrate surface;

FIGS. 7A through 7G are schematic, cross-sectional view which togetherdepict an example of a method for making an example flow cell includingan example of the first region in the depression and the second regionon the substrate surface;

FIGS. 8A through 8F are schematic, cross-sectional view which togetherdepict another example of a method for making another example flow cellincluding an example of the first region in the depression and thesecond region on the substrate surface;

FIG. 9 is a schematic, cross-sectional view of an example of the firstregion in the depression and the second region as part of a bead that ispositioned in the depression;

FIG. 10 is a schematic, cross-sectional view of an example of first andsecond primer sets attached to separate substrates;

FIGS. 11A through 11C are schematic perspective views which togetherdepict the formation of one example of a patterned resin on a support;

FIGS. 11A, 11B, and 11D are schematic perspective views which togetherdepict the formation of another example of a patterned resin on asupport;

FIG. 12 is a flow diagram illustrating an example of a method disclosedherein;

FIGS. 13A through 13D are schematic perspective views which togetherdepict an example of the method disclosed herein using the patternedsubstrate of FIG. 11C;

FIG. 14 is a cross-sectional view taken along line 4-4 of FIG. 13D;

FIGS. 15A through 15D are schematic perspective views which togetherdepict another example of the method disclosed herein using thepatterned substrate of FIG. 11C;

FIG. 16 is a cross-sectional view taken along line 6-6 of FIG. 15D;

FIGS. 17A through 17D are schematic perspective views which togetherdepict an example of the method disclosed herein using the patternedsubstrate of FIG. 11D;

FIG. 18 is a cross-sectional view taken along line 8-8 of FIG. 17D;

FIGS. 19A through 19D are schematic perspective views which togetherdepict another example of the method disclosed herein using thepatterned substrate of FIG. 11D;

FIG. 20 is a cross-sectional view taken along line 10-10 of FIG. 19D;

FIG. 21 is a flow diagram illustrating another example of a methoddisclosed herein;

FIGS. 22A through 22E are schematic perspective views which togetherdepict an example of the other method disclosed herein;

FIG. 22F is an enlarged view of a depression of an example of a flowcell shown in 22E, wherein the depression includes a block copolymer anda primer grafted to one block;

FIGS. 23A and 23B are schematic, top views of examples of depressionsand surrounding interstitial regions, where different examples of blockcopolymers are self-assembled and phase separated in the depressions,where each figure shows a different pattern of the blocks;

FIG. 24 depicts a schematic perspective view of an example of a flowcell disclosed herein;

FIGS. 25A and 25B are schematic cross-sectional views depictingdifferent examples of the flow cells disclosed herein which includetriblock copolymers;

FIGS. 26A through 26H are schematic views which together illustrate anexample method for forming the example regions shown in FIG. 2;

FIGS. 27A through 27F are schematic views which together illustrateanother example method for forming the example regions shown in FIG. 2;

FIGS. 28A through 27G are schematic views which together illustrate anexample method for forming the example regions shown in FIGS. 3A and 3B;

FIGS. 29A through 29H are schematic views which together illustrateanother example method for forming the example regions shown in FIGS. 3Aand 3B;

FIGS. 30A through 30F are schematic views which together illustratestill example method for forming the example regions shown in FIGS. 3Aand 3B;

FIGS. 31A through 31I are schematic views which together illustrate anexample method for forming the example regions shown in FIGS. 3A and 3B;

FIGS. 32A through 32F are schematic views which together illustrate anexample method for forming the example regions shown in FIG. 4C;

FIG. 33A is a schematic view of an example method for forming anotherexample of the regions disclosed herein;

FIG. 33B is a top view of one of the depressions of FIG. 33A,illustrating the regions;

FIGS. 34A through 34S are schematic views which together illustrate anexample method for forming the example regions in trenches; and

FIG. 35 schematically illustrates an example of functionalized supportstructures;

FIGS. 36A and 36B are schematic views which together illustrate anexample method for forming the example regions using the functionalizedsupport structures of FIG. 35;

FIGS. 37A and 37B are schematic views which together illustrate anexample method for forming the example regions using the functionalizedsupport structures of FIG. 35;

FIGS. 38A and 38B are schematic views which together illustrate anexample method involving another example of a block copolymer;

FIG. 39A is a scanning electron microscopy (SEM) image of a flow cellsubstrate having 50 μm diameter depressions separated by interstitialregions, with a pre-grafted polymer layer deposited thereon;

FIG. 39B is a SEM image of the flow cell substrate of FIG. 39A with aprotection layer deposited on the pre-grafted polymer layer;

FIG. 39C is a SEM image of the flow cell substrate of FIG. 39B afteretching to remove the protection layer from the interstitial regions;

FIGS. 40A and 40B depict the data analysis for a simultaneous paired endread (FIG. 40A) and a sequential paired end read (FIG. 40B);

FIGS. 41A through 41G are schematic views which together illustrateanother example method for forming the example regions shown in FIG. 2;and

FIGS. 42A through 42H are schematic views which together illustrate yetanother example method for forming the example regions shown in FIG. 2.

DETAILED DESCRIPTION

Examples of the flow cells disclosed herein may be used for nucleic acidsequencing.

Some of the flow cells include different primer sets attached todifferent regions of the flow cell substrate. In these examples, theprimer sets may be controlled so that the cleaving (linearization)chemistry is orthogonal in the different regions. Orthogonal cleavingchemistry may be realized through identical cleavage sites that areattached to different primers in the different sets, or throughdifferent cleavage sites that are attached to different primers in thedifferent sets. This enables a cluster of forward strands to begenerated in one region of the substrate and a cluster of reversestrands to be generated in another region of the substrate. In anexample, the regions are directly adjacent to one another. In anotherexample, any space between the regions is small enough that clusteringcan span the two regions. With some of the flow cell configurationsdisclosed herein, the forward and reverse strands are spatiallyseparate, which separates the fluorescence signals from both reads whileallowing for simultaneous base calling of each read. As such, someexamples of the flow cells disclosed herein enable simultaneouspaired-end reads to be obtained.

Other examples of the flow cells may be used to obtain simultaneouspaired-end reads; or may be used to obtain sequential paired-end reads,where the forward strands are sequenced and removed, and then thereverse strands are sequenced and removed. In these other examples, apatterned resin on a flow cell support is coated with a block copolymerthat undergoes directed self-assembly in depressions of the patternedresin. The patterned resin serves as a guide for the arrangement of theblock copolymer. Under controlled conditions, the block copolymerself-assembles into specific domains. In some of the examples disclosedherein, the functionality of the domains is controlled to be orthogonalso that one or more domains can react with the patterned resin and oneor more other domains can graft primer(s). In some examples, thefunctionality of a domain may be controlled to alter a characteristic ofthat domain. These example flow cells may be suitable for use withoptical or non-optical detection methods.

Definitions

It is to be understood that terms used herein will take on theirordinary meaning in the relevant art unless specified otherwise. Severalterms used herein and their meanings are set forth below.

The singular forms “a”, “an”, and “the” include plural referents unlessthe context clearly dictates otherwise.

The terms comprising, including, containing and various forms of theseterms are synonymous with each other and are meant to be equally broad.

The terms top, bottom, lower, upper, on, etc. are used herein todescribe the flow cell and/or the various components of the flow cell.It is to be understood that these directional terms are not meant toimply a specific orientation, but are used to designate relativeorientation between components. The use of directional terms should notbe interpreted to limit the examples disclosed herein to any specificorientation(s).

An “acrylamide monomer” is a monomer with the structure

or a monomer including an acrylamide group with that structure. Examplesof the monomer including an acrylamide group include azido acetamidopentyl acrylamide:

and N-isopropylacrylamide:

Other acrylamide monomers may be used.

An aldehyde, as used herein, is an organic compound containing afunctional group with the structure —CHO, which includes a carbonylcenter (i.e., a carbon double-bonded to oxygen) with the carbon atomalso bonded to hydrogen and an R group, such as an alkyl or other sidechain. The general structure of an aldehyde is:

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that is fully saturated (i.e., contains no double or triplebonds). The alkyl group may have 1 to 20 carbon atoms. Example alkylgroups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,tertiary butyl, pentyl, hexyl, and the like. As an example, thedesignation “C1-4 alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from thegroup consisting of methyl, ethyl, propyl, iso-propyl, n-butyl,isobutyl, sec-butyl, and t-butyl.

As used herein, “alkenyl” refers to a straight or branched hydrocarbonchain containing one or more double bonds. The alkenyl group may have 2to 20 carbon atoms. Example alkenyl groups include ethenyl, propenyl,butenyl, pentenyl, hexenyl, and the like.

As used herein, “alkyne” or “alkynyl” refers to a straight or branchedhydrocarbon chain containing one or more triple bonds. The alkynyl groupmay have 2 to 20 carbon atoms.

As used herein, “aryl” refers to an aromatic ring or ring system (i.e.,two or more fused rings that share two adjacent carbon atoms) containingonly carbon in the ring backbone. When the aryl is a ring system, everyring in the system is aromatic. The aryl group may have 6 to 18 carbonatoms. Examples of aryl groups include phenyl, naphthyl, azulenyl, andanthracenyl.

An “amino” functional group refers to an —NR_(a)R_(b) group, where R_(a)and R_(b) are each independently selected from hydrogen

C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-10 aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

As used herein, the term “attached” refers to the state of two thingsbeing joined, fastened, adhered, connected or bound to each other,either directly or indirectly. For example, a bead that is attached to adepression may be physically entrapped in the depression. For anotherexample, a nucleic acid can be attached to a functionalized polymer by acovalent or non-covalent bond. A covalent bond is characterized by thesharing of pairs of electrons between atoms. A non-covalent bond is aphysical bond that does not involve the sharing of pairs of electronsand can include, for example, hydrogen bonds, ionic bonds, van der Waalsforces, hydrophilic interactions and hydrophobic interactions. For stillanother example, a primer can be attached to a region on the substratethrough a support structure.

An “azide” or “azido” functional group refers to —N₃.

The terms “bead” or “core structure” of a functionalized bead refers toa small body made of a rigid or semi-rigid material. The body can have ashape characterized, for example, as a sphere, oval, microsphere, orother recognized particle shape whether having regular or irregulardimensions. Example materials that are useful for beads/core structuresinclude, without limitation, glass; plastic such as acrylic, polystyreneor a copolymer of styrene and another material, polypropylene,polyethylene, polybutylene, polyurethane or polytetrafluoroethylene(TEFLON®, from Chemours); polysaccharides or cross-linkedpolysaccharides such as agarose or Sepharose; nylon; nitrocellulose;resin; silica or silica-based materials including silicon and modifiedsilicon; carbon-fiber, metal; inorganic glass; optical fiber bundle, ora variety of other polymers. Example beads/core structures includecontrolled pore glass beads, paramagnetic beads, thoria sol, Sepharosebeads, nanocrystals and others known in the art as described, forexample, in Microsphere Detection Guide from Bangs Laboratories, FishersInd. Beads may also be coated with a polymer that has a functional groupthat can attach to a primer.

A “block copolymer” is a copolymer formed when two or more monomerscluster together and form blocks of repeating units. Each block shouldhave at least one feature and/or at least one block-specific functionalgroup which is/are not present in adjacent blocks. In the examplesdisclosed herein, the block copolymers are capable, when exposed toparticular annealing conditions, to self-assemble into ordered domainsat nanometer-scale dimensions by microphase separation of theconstituent polymer blocks. Specific examples of block copolymers willbe described further below.

A “block-specific functional group” refers to a moiety of atoms and/orbonds within a particular block of the block polymer that has aparticular functionality, such as reacting with a patterned resin,attaching a primer, adjusting an interaction parameter to drivemicrophase separation, altering a characteristic of the block copolymer,etc. In some examples disclosed herein, each block includes a differentblock-specific functional group. Specific examples of eachblock-specific functional group will be described further below.

As used herein, a “bonding region” refers to an area on a support thatis to be bonded to another material, which may be, as examples, a spacerlayer, a lid, another substrate, etc., or combinations thereof (e.g., aspacer layer and a lid). The bond that is formed at the bonding regionmay be a chemical bond (as described above), or a mechanical bond (e.g.,using a fastener, etc.).

A “capture site”, as used herein, refers to portion of a flow cellsurface having been physically modified and/or modified with a chemicalproperty that allows for localization of a functionalized supportstructure. In an example, the capture site may include a chemicalcapture agent

As used herein, “carbocyclyl” means a non-aromatic cyclic ring or ringsystem containing only carbon atoms in the ring system backbone. Whenthe carbocyclyl is a ring system, two or more rings may be joinedtogether in a fused, bridged or spiro-connected fashion. Carbocyclylsmay have any degree of saturation, provided that at least one ring in aring system is not aromatic. Thus, carbocyclyls include cycloalkyls,cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20carbon atoms. Examples of carbocyclyl rings include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene,bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

As used herein, the term “carboxylic acid” or “carboxyl” as used hereinrefers to —COOH.

A “chemical capture agent” is a material, molecule or moiety that iscapable of attaching, retaining, or binding to a target molecule (e.g.,a functionalized support structure). One example chemical capture agentincludes a capture nucleic acid (e.g., a capture oligonucleotide) thatis complementary to at least a portion of a target nucleic acid of orattached to the target molecule. Still another example chemical captureagent includes a member of a receptor-ligand binding pair (e.g., avidin,streptavidin, biotin, lectin, carbohydrate, nucleic acid bindingprotein, epitope, antibody, etc.) that is capable of binding to thetarget molecule (or to a linking moiety attached to the targetmolecule). Yet another example of the chemical capture agent is achemical reagent capable of forming an electrostatic interaction, ahydrogen bond, or a covalent bond (e.g., thiol-disulfide exchange, clickchemistry, Diels-Alder, etc.) with the target molecule.

As used herein, “cycloalkylene” means a fully saturated carbocyclyl ringor ring system that is attached to the rest of the molecule via twopoints of attachment.

As used herein, “cycloalkenyl” or “cycloalkene” means a carbocyclyl ringor ring system having at least one double bond, wherein no ring in thering system is aromatic. Examples include cyclohexenyl or cyclohexeneand norbornenyl or norbornene. Also as used herein, “heterocycloalkenyl”or “heterocycloalkene” means a carbocyclyl ring or ring system with atleast one heteroatom in ring backbone, having at least one double bond,wherein no ring in the ring system is aromatic.

As used herein, “cycloalkynyl” or “cycloalkyne” means a carbocyclyl ringor ring system having at least one triple bond, wherein no ring in thering system is aromatic. An example is cyclooctyne. Another example isbicyclononyne. Also as used herein, “heterocycloalkynyl” or“heterocycloalkyne” means a carbocyclyl ring or ring system with atleast one heteroatom in ring backbone, having at least one triple bond,wherein no ring in the ring system is aromatic.

The term “depositing,” as used herein, refers to any suitableapplication technique, which may be manual or automated, and, in someinstances, results in modification of the surface properties. Generally,depositing may be performed using vapor deposition techniques, coatingtechniques, grafting techniques, or the like. Some specific examplesinclude chemical vapor deposition (CVD), spray coating (e.g., ultrasonicspray coating), spin coating, dunk or dip coating, doctor blade coating,puddle dispensing, flow through coating, aerosol printing, screenprinting, microcontact printing, inkjet printing, or the like.

As used herein, the term “depression” refers to a discrete concavefeature in a substrate or a patterned resin having a surface openingthat is at least partially surrounded by interstitial region(s) of thesubstrate or the patterned resin. Depressions can have any of a varietyof shapes at their opening in a surface including, as examples, round,elliptical, square, polygonal, star shaped (with any number ofvertices), etc. The cross-section of a depression taken orthogonallywith the surface can be curved, square, polygonal, hyperbolic, conical,angular, etc. As examples, the depression can be a well or twointerconnected wells. The depression may also have more complexarchitectures, such as ridges, step features, etc.

The term “each,” when used in reference to a collection of items, isintended to identify an individual item in the collection, but does notnecessarily refer to every item in the collection. Exceptions can occurif explicit disclosure or context clearly dictates otherwise.

The term “epoxy” (also referred to as a glycidyl or oxirane group) asused herein refers to

As used herein, the term “flow cell” is intended to mean a vessel havinga chamber (i.e., flow channel) where a reaction can be carried out, aninlet for delivering reagent(s) to the chamber, and an outlet forremoving reagent(s) from the chamber. In some examples, the chamberenables the detection of the reaction that occurs in the chamber. Forexample, the chamber/flow channel can include one or more transparentsurfaces allowing for the optical detection of arrays, optically labeledmolecules, or the like, at the depression.

As used herein, a “flow channel” may be an area defined between twobonded components, which can selectively receive a liquid sample. Insome examples, the flow channel may be defined between a patterned resinand a lid, and thus may be in fluid communication with one or moredepressions defined in the patterned resin.

A “functionalized support structure” refers to a small body made of arigid or semi-rigid material that has one of the primer sets disclosedherein attached to its surface. The body can have a shape characterized,for example, as a sphere, oval, microsphere, or other recognizedparticle shape whether having regular or irregular dimensions. Examplematerials that are useful for the body include, without limitation,glass; plastic such as acrylic, polystyrene or a copolymer of styreneand another material, polypropylene, polyethylene, polybutylene,polyurethane or polytetrafluoroethylene (TEFLON®, from Chemours);polysaccharides or cross-linked polysaccharides such as agarose orSepharose; nylon; nitrocellulose; resin; silica or silica-basedmaterials including silicon and modified silicon; carbon-fiber, metal;inorganic glass; optical fiber bundle, or a variety of other polymers.

As used herein, “heteroaryl” refers to an aromatic ring or ring system(i.e., two or more fused rings that share two adjacent atoms) thatcontain(s) one or more heteroatoms, that is, an element other thancarbon, including but not limited to, nitrogen, oxygen and sulfur, inthe ring backbone. When the heteroaryl is a ring system, every ring inthe system is aromatic. The heteroaryl group may have 5-18 ring members.

As used herein, “heterocyclyl” means a non-aromatic cyclic ring or ringsystem containing at least one heteroatom in the ring backbone.Heterocyclyls may be joined together in a fused, bridged orspiro-connected fashion. Heterocyclyls may have any degree of saturationprovided that at least one ring in the ring system is not aromatic. Inthe ring system, the heteroatom(s) may be present in either anon-aromatic or aromatic ring. The heterocyclyl group may have 3 to 20ring members (i.e., the number of atoms making up the ring backbone,including carbon atoms and heteroatoms). In some examples, theheteroatom(s) are O, N, or S.

The term “hydrazine” or “hydrazinyl” as used herein refers to a —NHNH₂group.

As used herein, the term “hydrazone” or “hydrazonyl” as used hereinrefers to a

group in which R_(a) and R_(b) are each independently selected fromhydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl,C6-10 aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

As used herein, “hydroxy” or “hydroxyl” refers to an —OH group.

As used herein, the term “interstitial region” refers to an area, e.g.,of a substrate, patterned resin, or other support that separatesdepressions. For example, an interstitial region can separate onedepression of an array from another depression of the array. The twodepressions that are separated from each other can be discrete, i.e.,lacking physical contact with each other. In many examples, theinterstitial region is continuous whereas the depressions are discrete,for example, as is the case for a plurality of depressions defined in anotherwise continuous surface. In other examples, the interstitialregions and the features are discrete, for example, as is the case for aplurality of trenches separated by respective interstitial regions. Theseparation provided by an interstitial region can be partial or fullseparation. Interstitial regions may have a surface material thatdiffers from the surface material of the depressions defined in thesurface. For example, depressions can have a polymer and a first primerset therein, and the interstitial regions can have a polymer and asecond primer set thereon. For another example, depressions of an arraycan have beads therein while the interstitial regions do not have beadsthereon.

“Nitrile oxide,” as used herein, means a “R_(a)C≡N⁺O⁻” group in whichR_(a) is defined herein. Examples of preparing nitrile oxide include insitu generation from aldoximes by treatment with chloramide-T or throughaction of base on imidoyl chlorides [RC(Cl)═NOH] or from the reactionbetween hydroxylamine and an aldehyde.

“Nitrone,” as used herein, means a

group in which R₁, R₂, and R₃ may be any of the R_(a) and R_(b) groupsdefined herein.

As used herein, a “nucleotide” includes a nitrogen containingheterocyclic base, a sugar, and one or more phosphate groups.Nucleotides are monomeric units of a nucleic acid sequence. In RNA, thesugar is a ribose, and in DNA, the sugar is a deoxyribose, i.e. a sugarlacking a hydroxyl group that is present at the 2′ position in ribose.The nitrogen containing heterocyclic base (i.e., nucleobase) can be apurine base or a pyrimidine base. Purine bases include adenine (A) andguanine (G), and modified derivatives or analogs thereof. Pyrimidinebases include cytosine (C), thymine (T), and uracil (U), and modifiedderivatives or analogs thereof. The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine. A nucleic acid analog may haveany of the phosphate backbone, the sugar, or the nucleobase altered.Examples of nucleic acid analogs include, for example, universal basesor phosphate-sugar backbone analogs, such as peptide nucleic acid (PNA).

A “patterned resin” refers to any polymer that can have depressionsdefined therein. Specific examples of resins and techniques forpatterning the resins will be described further below. In some examplesdisclosed herein, the patterned resin can serve as a guiding templatefor a block copolymer to self-assemble thereon. Specific examples ofcharacteristics that render a polymer a “guiding template” will bedescribed further below.

As used herein, the “primer” is defined as a single stranded nucleicacid sequence (e.g., single strand DNA or single strand RNA). Someprimers, referred to herein as amplification primers, serve as astarting point for template amplification and cluster generation. Otherprimers, referred to herein as sequencing primers, serve as a startingpoint for DNA or RNA synthesis. The 5′ terminus of the primer may bemodified to allow a coupling reaction with a functional group of apolymer or with a bead surface. The primer length can be any number ofbases long and can include a variety of non-natural nucleotides. In anexample, the sequencing primer is a short strand, ranging from 10 to 60bases, or from 20 to 40 bases.

A “spacer layer,” as used herein refers to a material that bonds twocomponents together. In some examples, the spacer layer can be aradiation-absorbing material that aids in bonding, or can be put intocontact with a radiation-absorbing material that aids in bonding.

“Solvent annealing” or “solvent vapor annealing” involves exposing apolymer (e.g., in the form of a film or layer) to an excess of solventin a sealed enclosure to generate a saturated vapor (i.e., a solventatmosphere) above the polymer. The polymer film or layer may be held atroom temperature (e.g., from about 18° C. to about 25° C.) or at anelevated temperature, which causes the polymer to swell, and increaseits chain mobility.

The term “substrate” refers to a structure upon which various componentsof the flow cell (e.g., a polymer, primer(s), etc.) may be added. Thesubstrate may be a wafer, a panel, a rectangular sheet, a die, or anyother suitable configuration. The substrate is generally rigid and isinsoluble in an aqueous liquid. The substrate may be inert to achemistry that is used to modify the depressions or that is present inthe depressions. For example, a substrate can be inert to chemistry usedto form the polymer, to attach the primer(s), etc. The substrate may bea single layer structure, or a multi-layered structure (e.g., includinga support and a patterned resin on the support). Examples of suitablesubstrates will be described further below.

A “thiol” functional group refers to —SH.

As used herein, the terms “tetrazine” and “tetrazinyl” refer tosix-membered heteroaryl group comprising four nitrogen atoms. Tetrazinecan be optionally substituted.

“Tetrazole,” as used herein, refer to five-membered heterocyclic groupincluding four nitrogen atoms. Tetrazole can be optionally substituted.

Flow Cells for Simultaneous Paired-End Read Sequencing

An example of the flow cell disclosed herein includes a substrate; afirst primer set attached to a first region on the substrate, the firstprimer set including an un-cleavable first primer and a cleavable secondprimer; and a second primer set attached to a second region on thesubstrate, the second primer set including a cleavable first primer andan un-cleavable second primer.

Examples of suitable substrates include epoxy siloxane, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes,polytetrafluoroethylene (such as TEFLON® from Chemours), cyclicolefins/cyclo-olefin polymers (COP) (such as ZEONOR® from Zeon),polyimides, etc.), nylon, ceramics/ceramic oxides, silica, fused silica,or silica-based materials, aluminum silicate, silicon and modifiedsilicon (e.g., boron doped p+ silicon), silicon nitride (Si₃N₄), siliconoxide (SiO₂), tantalum pentoxide (Ta₂O₅) or other tantalum oxide(s)(TaO_(x)), hafnium oxide (HaO₂), carbon, metals, inorganic glasses, orthe like. The substrate may also be a multi-layered structure. Someexamples of the multi-layered structure include glass or silicon, with acoating layer of tantalum oxide or another ceramic oxide at the surface.Other examples of the multi-layered structure include an underlyingsupport (e.g., glass or silicon) having a patterned resin thereon. Stillother examples of the multi-layered substrate may include asilicon-on-insulator (SOI) substrate.

In an example, the substrate may have a diameter ranging from about 2 mmto about 300 mm, or a rectangular sheet or panel having its largestdimension up to about 10 feet (˜3 meters). In an example, the substrateis a wafer having a diameter ranging from about 200 mm to about 300 mm.In another example, the substrate is a die having a width ranging fromabout 0.1 mm to about 10 mm. While example dimensions have beenprovided, it is to be understood that a substrate with any suitabledimensions may be used. For another example, a panel may be used that isa rectangular support, which has a greater surface area than a 300 mmround wafer.

In some examples of the flow cell, a first primer set is attached to afirst region on the substrate and a second primer set is attached to asecond region on the substrate. FIG. 1A through FIG. 1D depict differentconfigurations of the primer sets 12A, 12A′, 12B, 12B′, 12C, 12C′, and12D, 12D′ attached to the different regions 14, 16.

Each of the first primer sets 12A, 12B, 12C, and 12D includes anun-cleavable first primer 18 or 18′ and a cleavable second primer 20 or20′; and each of the second primer sets 12A′, 12B′, 12C′, and 12D′includes a cleavable first primer 19 and an un-cleavable second primer21.

The un-cleavable first primer 18 or 18′ and the cleavable second primer20 or 20′ are oligo pairs, e.g., where the un-cleavable first primer 18or 18′ is a forward amplification primer and the cleavable second primer20 or 20′ is a reverse amplification primer or where the cleavablesecond primer 20 or 20′ is the forward amplification primer and theun-cleavable first primer 18 or 18′ is the reverse amplification primer.In each example of the first primer set 12A, 12B, 12C, and 12D, thecleavable second primer 20 or 20′ includes a cleavage site 22, while theun-cleavable first primer 18 or 18′ does not include a cleavage site 22.

The cleavable first primer 19 or 19′ and the un-cleavable second primer21 or 21′ are also oligo pairs, e.g., where the cleavable first primer19 or 19′ is a forward amplification primer and un-cleavable secondprimer 21 or 21′ is a reverse amplification primer or where theun-cleavable second primer 21 or 21′ is the forward amplification primerand the cleavable first primer 19 or 19′ is the reverse amplificationprimer. In each example of the second primer set 12A′, 12B′, 12C′, and12D′, the cleavable first primer 19 or 19′ includes a cleavage site 22′or 23, while the un-cleavable second primer 21 or 21′ does not include acleavage site 22′ or 23.

It is to be understood that the un-cleavable first primer 18 or 18′ ofthe first primer set 12A, 12B, 12C, 12D and the cleavable first primer19 or 19′ of the second primer set 12A′, 12B′, 12C′, and 12D′ have thesame nucleotide sequence (e.g., both are forward amplification primers),except that the cleavable first primer 19 or 19′ includes the cleavagesite 22′ or 23 integrated into the nucleotide sequence or into a linker24′ attached to the nucleotide sequence. Similarly, the cleavable secondprimer 20 or 20′ of the first primer set 12A, 12B, 12C, 12D and theun-cleavable second primer 21 or 21′ of the second primer set 12A′,12B′, 12C′, and 12D′ have the same nucleotide sequence (e.g., both arereverse amplification primers), except that the cleavable second primer20 or 20′ includes the cleavage site 22 integrated into the nucleotidesequence or into a linker 24 attached to the nucleotide sequence.

It is to be understood that when the first primers 18 and 19 or 18′ and19′ are forward amplification primers, the second primers 20 and 21 or20′ and 21′ are reverse primers, and vice versa.

Examples of un-cleavable primers 18, 21 or 18′, 21′ include P5 and P7primers, examples of which are used on the surface of commercial flowcells sold by Illumina Inc. for sequencing, for example, on HISEQ™,HISEQX™, MISEQ™, MISEQDX™, MINISEQ™, NEXTSEQ™, NEXTSEQDX™, NOVASEQ™,ISEQ™, GENOME ANALYZER™, and other instrument platforms. The P5 and P7primers have a universal sequence for capture and/or amplificationpurposes. In an example, the P5 and P7 primers include the following:

P5: 5′ → 3′ (SEQ. ID. NO. 1) AATGATACGGCGACCACCGA P7: 5′ → 3′(SEQ. ID. NO. 2) CAAGCAGAAGACGGCATACGAThe P5 and P7 primers are un-cleavable primers 18, 21 or 18′, 21′because they do not include a cleavage site 22, 22′, 23. It is to beunderstood that any suitable universal sequence can be used as theun-cleavable primers 18, 21 or 18′, 21′.

Examples of cleavable primers 19, 20 or 19′, 20′ include the P5 and P7(or other universal sequence) primers with the respective cleavage sites22, 22′, 23 incorporated into the respective nucleic acid sequences(e.g., FIG. 1A and FIG. 1C), or into a linker 24′, 24 that attaches thecleavable primers 19, 20 or 19′, 20′ to the respective regions 16, 14(FIG. 1B and FIG. 1D). Examples of suitable cleavage sites 22, 22′, 23include enzymatically cleavable nucleobases or chemically cleavablenucleobases, modified nucleobases, or linkers (e.g., betweennucleobases). The enzymatically cleavable nucleobase may be susceptibleto cleavage by reaction with a glycosylase and an endonuclease, or withan exonuclease. One specific example of the cleavable nucleobase isdeoxyuracil (dU), which can be targeted by the USER enzyme. In anexample, the uracil base may be incorporated at the 7^(th) base positionfrom the 3′ end of the P5 primer (P5U) or of the P7 primer (P7U). Otherabasic sites may also be used. Examples of the chemically cleavablenucleobases, modified nucleobases, or linkers include a vicinal diol, adisulfide, a silane, an azobenzene, a photocleavable group, allyl T (athymine nucleotide analog having an allyl functionality), allyl ethers,or an azido functional ether.

Each primer set 12A and 12A′ or 12B and 12B′ or 12C and 12C′ or 12D and12D′ is attached to a respective region 14 or 16 on the substrate. Insome examples, the regions 14, 16 have the same surface chemistry, andany of the techniques set forth herein may be used to graft one set ofprimers 18, 20 or 18′, 20′ on the region 14, and another set of primers19, 21 or 19′, 21′ on the region 16. In other examples, the regions 14or 16 include different surface chemistries (e.g., functional groups)that can selectively react with the respective primers 18, 20 or 18′,20′ or 19, 21 or 19′, 21′. In these other examples, the first region 14has a first functional group, and the second region 16 has a secondfunctional group that is different than the first functional group.

As mentioned, FIG. 1A through FIG. 1D depict different configurations ofthe primer sets 12A, 12A′, 12B, 12B′, 12C, 12C′, and 12D, 12D′ attachedto the different regions 14, 16. More specifically, FIG. 1A through FIG.1D depict different configurations of the primers 18, 20 and 19, 21 or18′, 20′ and 19′, 21′ that may be used.

In the example shown in FIG. 1A, the primers 18, 20 and 19, 21 of theprimer sets 12A and 12A′ are directly attached to the regions 14 and 16,for example, without a linker 24, 24′. The region 14 may have surfacefunctional groups that can immobilize the terminal groups at the 5′ endof the primers 18, 20. Similarly, the region 16 may have surfacefunctional groups that can immobilize the terminal groups at the 5′ endof the primers 19, 21. In one example, the immobilization chemistrybetween the region 14 and the primers 18, 20 and the immobilizationchemistry between the region 16 and the primers 19, 21 may be differentso that the primers 18, 20 or 19, 21 selectively attach to the desirableregion 14 or 16. In another example, the immobilization chemistry may bethe same for the regions 14, 16 and the respective primers 18, 20 or 19,21, and a patterning technique may be used to graft one primer set 12A,12A′ at a time. In still another example, the materials applied to formthe regions 14, 16 may have the respective primers 18, 20 or 19, 21pre-grafted thereto, and thus the immobilization chemistries may be thesame or different.

In this example, immobilization may be by single point covalentattachment to the respective region 14 or 16 at the 5′ end of therespective primers 18 and 20 or 19 and 21. Any suitable covalentattachment means known in the art may be used at the regions 14, 16.Examples of terminated primers that may be used include an alkyneterminated primer, a tetrazine terminated primer, an azido terminatedprimer, an amino terminated primer, an epoxy or glycidyl terminatedprimer, a thiophosphate terminated primer, a thiol terminated primer, analdehyde terminated primer, a hydrazine terminated primer, aphosphoramidite terminated primer, and a triazolinedione terminatedprimer. In some specific examples, a succinimidyl (NHS) ester terminatedprimer may be reacted with an amine at a surface of the region 14 and/or16, an aldehyde terminated primer may be reacted with a hydrazine at asurface of the region 14 and/or 16, or an alkyne terminated primer maybe reacted with an azide at a surface of the region 14 and/or 16, or anazide terminated primer may be reacted with an alkyne or DBCO(dibenzocyclooctyne) at a surface of the region 14 and/or 16, or anamino terminated primer may be reacted with an activated carboxylategroup or NHS ester at a surface of the region 14 and/or 16, or a thiolterminated primer may be reacted with an alkylating reactant (e.g.,iodoacetamine or maleimide) at a surface of the region 14 and/or 16, aphosphoramidite terminated primer may be reacted with a thioether at asurface of the region 14 and/or 16, or a biotin-modified primer may bereacted with streptavidin at a surface of the region 14 and/or 16.

Also in the example shown in FIG. 1A, the cleavage site 22, 22′ of eachof the cleavable primers 20, 19 is incorporated into the sequence of theprimer 20, 19. In this example, the same type of cleavage site 22, 22′is used in the cleavable primers 20, 19 of the respective primer sets12A, 12A′. As an example, the cleavage sites 22, 22′ are uracil bases,and the cleavable primers 20, 19 are P5U and P7U. In this example, theun-cleavable primer 18 of the oligo pair 18, 20 may be P7, and theun-cleavable primer 21 of the oligo pair 19, 21 may be P5. Thus, in thisexample, the first primer set 12A includes P7, P5U and the second primerset 12A′ includes P5, P7U. The primer sets 12A, 12A′ have oppositelinearization chemistries, which, after amplification, clustergeneration, and linearization, enables forward template strands to beformed on one region 14 or 16 and reverse strands to be formed on theother region 16 or 14.

In the example shown in FIG. 1B, the primers 18′, 20′ and 19′, 21′ ofthe primer sets 12B and 12B′ are attached to the regions 14 and 16, forexample, through linkers 24, 24′. The region 14 may have surfacefunctional groups that can immobilize the linker 24 at the 5′ end of theprimers 18′, 20′. Similarly, the region 16 may have surface functionalgroups that can immobilize the linker 24′ at the 5′ end of the primers19′, 21′. In one example, the immobilization chemistry for the region 14and the linkers 24 and the immobilization chemistry for the region 16and the linkers 24′ may be different so that the primers 18′, 20′ or19′, 21′ selectively graft to the desirable region 14 or 16. In anotherexample, the immobilization chemistry may be the same for the regions14, 16 and the linkers 24, 24′, and any suitable technique disclosedherein may be used to graft one primer set 12B, 12B′ at a time. In stillanother example, the materials applied to form the regions 14, 16 mayhave the respective primers 18′, 20′ and 19′, 21′ pre-grafted thereto,and thus the immobilization chemistries may be the same or different.Examples of suitable linkers 24, 24′ may include nucleic acid linkers(e.g., 10 nucleotides or less) or non-nucleic acid linkers, such as apolyethylene glycol chain, an alkyl group or a carbon chain, analiphatic linker with vicinal diols, a peptide linker, etc. An exampleof a nucleic acid linker is a polyT spacer, although other nucleotidescan also be used. In one example, the spacer is a 6T to 10T spacer. Thefollowing are some examples of nucleotides including non-nucleic acidlinkers (where B is the nucleobase and “oligo” is the primer):

In the example shown in FIG. 1B, the primers 18′, 19′ have the samesequence and the same or different linker 24, 24′. The primer 18′ inun-cleavable, whereas the primer 19′ includes the cleavage site 22′incorporated into the linker 24′. Also in this example, the primers 20′,21′ have the same sequence (e.g., P7) and the same or different linker24, 24′. The primer 21′ in un-cleavable, and the primer 20′ includes thecleavage site 22 incorporated into the linker 24. The same type ofcleavage site 22, 22′ is used in the linker 24, 24′ of each of thecleavable primers 20′, 19′. As an example, the cleavage sites 22, 22′may be uracil bases that are incorporated into nucleic acid linkers 24,24′. The primer sets 12B, 12B′ have opposite linearization chemistries,which, after amplification, cluster generation, and linearization,enables forward template strands to be formed on one region 14 or 16 andreverse strands to be formed on the other region 16 or 14.

The example shown in FIG. 1C is similar to the example shown in FIG. 1A,except that different types of cleavage sites 22, 23 are used in thecleavable primers 20, 19 of the respective primer sets 12C, 12C′. Asexamples, two different enzymatic cleavage sites may be used, twodifferent chemical cleavage sites may be used, or one enzymatic cleavagesite and one chemical cleavage site may be used. Examples of differentcleavage sites 22, 23 that may be used in the respective cleavableprimers 20, 19 include any combination of a vicinal diol, a uracil, anallyl ether, a disulfide, a restriction enzyme site, and 8-oxoguanine.

The example shown in FIG. 1D is similar to the example shown in FIG. 1B,except that different types of cleavage sites 22, 23 are used in thelinkers 24, 24′ attached to the cleavable primers 20′, 19′ of therespective primer sets 12D, 12D′. Examples of different cleavage sites22, 23 that may be used in the respective in the linkers 24, 24′attached to the cleavable primers 20, 19 include any combination of a avicinal diol, a uracil, an allyl ether, a disulfide, a restrictionenzyme site, and 8-oxoguanine.

In any of the examples shown in FIG. 1A through FIG. 1D, the attachmentof the primers 18, 20 and 19, 21 or 18′, 20′ and 19′, 21′ to the regions14, 16 leaves a template-specific portion of the primers 18, 20 and 19,21 or 18′, 20′ and 19′, 21′ free to anneal to its cognate template andthe 3′ hydroxyl group free for primer extension.

The regions 14, 16 represent different areas on the substrate that havedifferent primer sets 12A, 12A′, or 12B, 12B′, or 12C, 12C′, or 12D,12D′ attached thereto. The regions 14, 16 may include materials withdifferent functional groups. In some instances the different functionalgroups are surface functional groups of the substrate or functionalgroups that have been introduced to a surface of the substrate, or maybe functional groups of another component (e.g., a polymer layer, abead, etc.) that is deposited on the substrate.

In some examples, the regions 14, 16 are chemically the same, and anytechnique disclosed herein may be used to sequentially attach theprimers 18, 20 and 19, 21 or 18′, 20′ and 19′, 21′ of the respectivesets 12A and 12A′, or 12B and 12B′, or 12C and 12C′, or 12D and 12D′ tothe respective regions 14, 16.

In one example where the regions 14, 16 are chemically the same, theboth regions 14, 16 include the same polymer layer. The polymer layermay be a semi-rigid polymeric material that is permeable to liquids andgases. An example of the polymer layer includes an acrylamide copolymer,such as poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide, PAZAM.PAZAM and some other forms of the acrylamide copolymer are representedby the following structure (I):

wherein:

-   -   R^(A) is selected from the group consisting of azido, optionally        substituted amino, optionally substituted alkenyl, optionally        substituted hydrazone, optionally substituted hydrazine,        carboxyl, hydroxy, optionally substituted tetrazole, optionally        substituted tetrazine, nitrile oxide, nitrone, and thiol;    -   R^(B) is H or optionally substituted alkyl;    -   R^(C), R^(D), and R^(E) are each independently selected from the        group consisting of H and optionally substituted alkyl;    -   each of the —(CH₂)_(p)— can be optionally substituted;    -   p is an integer in the range of 1 to 50;    -   n is an integer in the range of 1 to 50,000; and    -   m is an integer in the range of 1 to 100,000.        One of ordinary skill in the art will recognize that the        arrangement of the recurring “n” and “m” features in        structure (I) are representative, and the monomeric subunits may        be present in any order in the polymer structure (e.g., random,        block, patterned, or a combination thereof).

The molecular weight of the PAZAM may range from about 10 kDa to about1500 kDa, or may be, in a specific example, about 312 kDa.

In some examples, PAZAM is a linear polymer. In some other examples,PAZAM is a lightly cross-linked polymer.

In other examples, the polymer 26 may be a variation of the structure(I). In one example, the acrylamide unit may be replaced withN,N-dimethylacrylamide (

In this example, the acrylamide unit in structure (I) may be replacedwith

where R^(D), R^(E), and R^(F) are each H, and R^(G) and R^(H) are each amethyl group (instead of H as is the case with the acrylamide). In thisexample, q may be an integer in the range of 1 to 100,000. In anotherexample, the N,N-dimethylacrylamide may be used in addition to theacrylamide unit. In this example, structure (I) may include

in addition to the recurring “n” and “m” features, where R^(D), R^(E),and R^(F) are each H, and R^(G) and R^(H) are each a methyl group. Inthis example, q may be an integer in the range of 1 to 100,000.

As another example polymer, the recurring “n” feature in structure (I)may be replaced with a monomer including a heterocyclic azido grouphaving structure (II):

wherein R¹ is H or C1-C4 alkyl; R₂ is H or C1-C4 alkyl; L is a linkerincluding a linear chain with 2 to 20 atoms selected from the groupconsisting of carbon, oxygen, and nitrogen and 10 optional substituentson the carbon and any nitrogen atoms in the chain; E is a linear chainincluding 1 to 4 atoms selected from the group consisting of carbon,oxygen and nitrogen, and optional substituents on the carbon and anynitrogen atoms in the chain; A is an N substituted amide with an H orC1-C4 alkyl attached to the N; and Z is a nitrogen containingheterocycle. Examples of Z include 5 to 10 ring members present as asingle cyclic structure or a fused structure.

As still another example, the polymer may include a recurring unit ofeach of structure (III) and (IV):

wherein each of R^(1a), R^(2a), R^(1b) and R^(2b) is independentlyselected from hydrogen, optionally substituted alkyl or optionallysubstituted phenyl; each R^(3a) and R^(3b) is independently selectedfrom hydrogen, optionally substituted alkyl, optionally substitutedphenyl, or optionally substituted C7-C14 aralkyl; and each L¹ and L² isindependently selected from an optionally substituted alkylene linker oran optionally substituted heteroalkylene linker.

It is to be understood that other molecules may be used to form thepolymer layer, as long as they are functionalized to interact with thefirst and second primer sets 12A, 12A′ or 12B, 12B′, or 12C, 12C′, or12D, 12D′. Other examples of suitable polymer layers include thosehaving a colloidal structure, such as agarose; or a polymer meshstructure, such as gelatin; or a cross-linked polymer structure, such aspolyacrylamide polymers and copolymers, silane free acrylamide (SFA), oran azidolyzed version of SFA. Examples of suitable polyacrylamidepolymers may be synthesized from acrylamide and an acrylic acid or anacrylic acid containing a vinyl group, or from monomers that form [2+2]photo-cycloaddition reactions. Still other examples of suitable polymerlayers include mixed copolymers of acrylamides and acrylates. Branchedpolymers, such as star polymers, star-shaped or star-block polymers,dendrimers, and the like may also be used.

In other examples, the regions 14, 16 are chemically different. Forexample, the region 14 may have surface functional groups that canimmobilize the primers 18, 20 or 18′, 20′ of the first primer sets 12A,12B, 12C, 12D, and the region 16 may have different surface functionalgroups that can immobilize the primers 19, 21 or 19′, 21′ of the secondprimer sets 12A′, 12B′, 12C′, 12D′.

In one example where the regions 14, 16 are chemically different, ablock copolymer is used. In this example, the block copolymer includestwo different blocks, one with primer-grafting functional groups thatcan attach to the primers 18, 20 or 18′, 20′ of the first primer sets12A, 12B, 12C, 12D and another with primer-grafting functional groupsthat can attach to the primers 19, 21 or 19′, 21′ of the second primersets 12A′, 12B′, 12C′, 12D′. Examples of primer-grafting functionalgroup are selected from the group consisting of azide/azido, optionallysubstituted amino, optionally substituted alkenyl, aldehyde, optionallysubstituted hydrazone, optionally substituted hydrazine, carboxyl,hydroxy, optionally substituted tetrazole, optionally substitutedtetrazine, nitrile oxide, nitrone, thiol, and combinations thereof.

Other examples of chemically different regions 14, 16 include gold andPAZAM, gold and aluminum, silanes having two different surfacefunctional groups (e.g., azides and amines), a thiol self-assembledmonolayer on gold and a phosphonate self-assembled monolayer on aluminumor hafnium oxide, SiO₂ and Ta₂O₅, epoxy and Ta₂O₅, a first polymerincluding azide groups and a second polymer including amine groups, SiO₂and copper, or epoxy and copper. While several examples have beenprovided, it is to be understood that other combinations of chemicallydifferent regions 14, 16 may be used.

The regions 14, 16 may also have different physical configurations. FIG.2 through FIG. 6B illustrate different examples of these configurations.In these examples, the substrate 26 is shown as a single layer/material,such as glass, silicon, etc. It is to be understood, however, that amulti-layered substrate may be used with any of these exampleconfigurations. For example, any of these examples may include a supportand a patterned resin formed on the support.

FIG. 2 illustrates an example where the regions 14, 16 are located ondifferent areas of a surface S of the substrate 26.

One example method for making the example shown in FIG. 2 is shown inFIG. 26A through FIG. 26H. While the primer sets 12A, 12A′ or 12B, 12B′or 12C, 12C′ or 12D, 12D′ are mentioned throughout this description,they are not shown for clarity.

As shown at FIG. 26A, a first functionalized layer 60 is applied on asubstrate 26. The first functionalized layer 60 may be a polymer(PAZAM), a silane, a metal (gold, aluminum, etc.) or any other materialthat has a functional group that can attach to the first primer set 12A,12B, 12C, 12D. The first functionalized layer 60 may be deposited usingany of the techniques described herein.

Depending upon the first functionalized layer 60 that is used, thesubstrate 26 may be activated using silanization or plasma ashing togenerate surface groups that can react with the first functionalizedlayer 60. Examples of silanization and plasma ashing are described inmore detail in reference to FIG. 13A. The first functionalized layer 60may then be deposited using any of the techniques described herein.Depending upon the material used, the first functionalized layer 60 mayalso be cured.

In FIG. 26B and FIG. 26C, the first functionalized layer 60 is thenpatterned to form a first functionalized region (region 14) covered by aphotoresist 62. In an example, the photoresist 62 is a negativephotoresist (exposed region becomes insoluble). An example of suitablenegative photoresist includes the epoxy-based SU-8 photoresist(available from MicroChemicals). The photoresist 62 is applied to thefirst functionalized layer 60, is selectively exposed to certainwavelengths of light to form the insoluble region (shown at 62), and isexposed to a developer solution to remove the soluble portions. Inanother example, the photoresist 62 is a positive photoresist (exposedregion becomes soluble). Examples of suitable positive photoresistsinclude the MICROPOSIT® S1800 series or the AZ® 1500 series, both ofwhich are available from MicroChemicals. The photoresist 62 is appliedto the substrate 26, is selectively exposed to certain wavelengths oflight to form the soluble region, and is exposed to a developer solutionto remove the soluble portions, leaving the insoluble region (shown at62). In other examples, the photoresist 62 may be replaced with ananoimprint lithography resin that is patterned to form the region(e.g., 62).

As shown in FIG. 26C, the exposed portions of the first functionalizedlayer 60 (e.g., those not covered by the photoresist 62) may then beremoved, e.g., via etching or another suitable technique.

As shown in FIG. 26D, the second functionalized layer 64 is thenapplied, using any suitable deposition technique, on the photoresist 62and on portions (e.g., the exposed surface S) of the substrate 26.Depending upon the material used, the second functionalized layer 64 mayalso be cured.

As shown in FIG. 26E, the photoresist 62 may then be lifted off, whichalso removes any of the second functionalized layer 64 thereon.

In FIG. 26F and FIG. 26G, a portion of the second functionalized layer64 is removed. To remove the portion(s), a second photoresist 62′ isapplied, exposed, and developed so that the insoluble region (shown at62′) covers the first functionalized region 14 and a desirable portionof the second functionalized layer 64 that is i) adjacent to the firstfunctionalized region 14 and ii) to become the second functionalizedregion 16. Once the photoresist 62′ is formed, the exposed portions ofthe second functionalized layer 64 (e.g., those not covered by thephotoresist 62′) may then be removed, e.g., via etching or anothersuitable technique.

As shown in FIG. 26H, the photoresist 62′ may then be lifted off, whichexposes the first and second functionalized regions 14, 16.

In some examples, the primers 18, 20 or 18′, 20′ (not shown in FIG. 26Athrough FIG. 26H) may be pre-grafted to the first functionalized layer60, and thus are attached to the first functionalized region 14.Similarly, the primers 19, 21 or 19′, 21′ (not shown in FIG. 26A throughFIG. 26H) may be pre-grafted to the second functionalized layer 64, andthus are attached to the second functionalized region 16. In theseexamples, additional primer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 26A). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 26H), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 26D). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 26H), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished by flow through deposition (e.g., using a temporarily boundlid), dunk coating, spray coating, puddle dispensing, or by anothersuitable method that will attach the primer(s) 18, 20 or 18′, 20′ to theregion 14 or that will attach the primer(s) 19, 21 or 19′, 21′ to theregion 16. Each of these example techniques may utilize a primersolution or mixture, which may include the primer(s) 18, 20 or 18′, 20′,19, 21 or 19′, 21′ water, a buffer, and a catalyst. With any of thegrafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the substrate26.

While not shown in FIG. 26A through FIG. 26H, this example method mayfurther include depositing a first self-assembled monolayer (SAM) on thefirst functionalized region 14 and depositing a second self-assembledmonolayer (SAM) on the second functionalized region 16. In an example,the region 14 is gold, and the first SAM includes thiol groups that canattach to the gold and azide groups that can attach to the primers 18,20 or 18′, 20′. In another example, the region 16 is hafnium oxide oraluminum oxide, and the second SAM includes phosphonate groups that canattach to the hafnium oxide or aluminum oxide and amine groups that canattach to the primers 19, 21 or 19′, 21′. In the example using theself-assembled monolayers, the primer sets 12A, 12A′ or 12B, 12B′ or12C, 12C′ or 12D, 12D′ are grafted after the SAMS are formed.

Another example method for making the example shown in FIG. 2 is shownin FIG. 27A through FIG. 27F. While the primer sets 12A, 12A′ or 12B,12B′ or 12C, 12C′ or 12D, 12D′ are mentioned throughout thisdescription, they are not shown for clarity.

As shown at FIG. 27A, a first photoresist 62 is applied on the substrate26 so that a first substrate portion 66 remains exposed. In thisexample, the photoresist 62 may be a positive photoresist (exposedregion becomes soluble) or a negative photoresist (exposed regionbecomes insoluble). The photoresist 62 may also be replaced with ananoimprint lithography resin that is patterned to form a region (e.g.,62).

As shown in FIG. 27B, the first functionalized layer 60 may be depositedon the photoresist 62 and on the first substrate portion 66 using any ofthe techniques described herein. In some instances, the firstfunctionalized layer 60 may also be cured.

As shown in FIG. 27C, the photoresist 62 may then be lifted off, whichalso removes any of the first functionalized layer 60 thereon. Thisleaves the region 14 formed on the substrate surface S.

A second photoresist 62′ is applied, exposed, and developed so that theinsoluble region (shown at 62′) covers the first functionalized region14 and the substrate 26, except at a second substrate portion 68 that isadjacent to the first functionalized region 14.

As shown in FIG. 27E, the second functionalized layer 64 is thenapplied, using any suitable deposition technique and, in some instancescuring, on the photoresist 62′ and on the second substrate portion 68.

As shown in FIG. 27F, the photoresist 62′ may then be lifted off, whichremoves any of the second functionalized layer 64 thereon. This exposesthe first and second functionalized regions 14, 16.

In some examples, the primers 18, 20 or 18′, 20′ (not shown in FIG. 27Athrough FIG. 27F) may be pre-grafted to the first functionalized layer60, and thus are attached to the first functionalized region 14.Similarly, the primers 19, 21 or 19′, 21′ (not shown in FIG. 27A throughFIG. 27F) may be pre-grafted to the second functionalized layer 64, andthus are attached to the second functionalized region 16. In theseexamples, additional primer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 27B or 27C). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 27F), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 27E). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 27F), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished using any grafting technique disclosed herein. With any ofthe grafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the substrate26.

While not shown in FIG. 27A through FIG. 27F, this example method mayfurther include depositing a first self-assembled monolayer (SAM) on thefirst functionalized region 14 and depositing a second self-assembledmonolayer (SAM) on the second functionalized region 16. In the exampleusing the self-assembled monolayers, the primer sets 12A, 12A′ or 12B,12B′ or 12C, 12C′ or 12D, 12D′ are grafted after the SAMS are formed.

Yet another example method for making the example shown in FIG. 2 isshown in FIG. 41A through FIG. 41G. While the primer sets 12A, 12A′ or12B, 12B′ or 12C, 12C′ or 12D, 12D′ are mentioned throughout thisdescription, they are not shown for clarity.

The substrate 26 may be any examples of the substrate disclosed herein.While not shown, it is to be understood that the substrate may also be amulti-layered substrate including an un-patterned resin on a support(e.g., a nanoimprint lithography resin on a glass support, or any otherexample of the resin 54 and support 52 described herein, see the section“Bead Based Flow Cell”).

As shown in FIG. 41A, in this example, a sacrificial metal layer 98 isapplied on the substrate 26. In an example, the nanoimprint lithographyresin/resist of the multi-layered substrate may be exposed to oxygenplasma, and then the sacrificial metal layer 98 may be deposited usingany suitable metal deposition technique. In an example, the sacrificialmetal layer 98 is deposited using sputtering. Examples of thesacrificial metal layer 98 include aluminum or copper, and the layer 98may have a thickness ranging from about 10 nm to about 100 nm.

As shown in FIG. 41B, a resist is applied to the sacrificial metal layer98 and is patterned to define a multi-level or multi-depth depressiontherein. The patterned resin is shown at reference numeral 54′. In thisexample, the resin may be any nanoimprint lithography resin. In anexample, the resin is spin coated and soft baked, and then stamped andcured (e.g., using ultraviolet curing) to define a multi-level ormulti-depth depression that includes a deep portion 70 and the shallowportion 72 which is defined, in part, by a step portion 74 of thepatterned resin 54′.

Wet or dry etching may then be used to expose a portion 100 of thesubstrate 26 (e.g., a portion of the un-patterned resin or glass of themulti-layered substrate) underlying the deep portion 70 and a portion102 of the sacrificial metal layer 98 underlying the shallow portion 72.The exposed portions are shown in FIG. 41C. In an example of wetetching, FeCl₃ may be used to remove a copper sacrificial metal layer98. In another example of wet etching, KOH may be used to remove analuminum sacrificial metal layer 98. In an example of dry etching,oxygen plasma may first be used to remove residue of the patterned resin54′, and then a combination of Cl₂ and BCl₃ plasmas may be used to etchan aluminum sacrificial metal layer 98. Oxygen plasma may again be usedto clean the exposed portions 100 and 102. In this example, the depthsD1 (e.g., of the shallow portion 72) and D2 (e.g., from the bottom ofthe deep portion 70 to the top of the step portion 74), and thethickness D3 of the sacrificial metal layer 98 may be the same orsimilar (e.g., within one nm of each other) so that the desiredthickness of each of the materials 54′ and 98 is removed during etchingin order to expose the portions 100 and 102.

As shown in FIG. 41D, the first functionalized layer 60 may be depositedon the remaining patterned resin 54′, on the exposed portion 100 of thesubstrate 26, and on the exposed portion 102 of the sacrificial metallayer 98. The first functionalized layer 60 may be any of the examplesand may be deposited using any of the techniques described herein. Insome instances, the first functionalized layer 60 is also cured.

In FIG. 41E, wet etching is used to selectively remove a portion of thefirst functionalized layer 60 and another portion of the sacrificialmetal layer 98 in order to expose another portion 104 of the substrate26. Wet etching may be performed as described herein. The etchant usedcan etch the sacrificial metal layer 98, thus lifting off the firstfunctional layer 60.

As shown in FIG. 41F, the second functionalized layer 64 is thenapplied, on the exposed portion 104 and on the first functionalizedlayer 60. Any suitable deposition technique may be used for the secondfunctionalized layer 64. In any of the example methods disclosed herein,when deposition is performed under high ionic strength (e.g., in thepresence of 10× PBS, NaCl, KCl, etc.), the second functionalized layer64 does not deposit on or adhere to the first functionalized layer 60.As such, the second functionalized layer 64 does not contaminate thefirst functionalized layer 60, leaving the region 16.

As shown in FIG. 41G, the remaining patterned resin 54 may then belifted off, which removes any of the first functionalized layer 60thereon. This lift off process may be performed in dimethylsulfoxide(DMSO) using sonication, or in acetone, or with an NMP(N-methyl-2-pyrrolidone) based stripper. The remaining sacrificial metallayer 98 is then exposed, and can be removed using wet etching asdescribed herein. The regions 14, 16 remain intact on the substratesurface after wet etching, in part because the sacrificial metal layer98 is not present under the regions 14, 16.

In some examples, the primers 18, 20 or 18′, 20′ (not shown in FIG. 41Athrough FIG. 41G) may be pre-grafted to the first functionalized layer60, and thus are attached to the first functionalized region 14.Similarly, the primers 19, 21 or 19′, 21′ (not shown in FIG. 41A throughFIG. 41G) may be pre-grafted to the second functionalized layer 64, andthus are attached to the second functionalized region 16. In theseexamples, additional primer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 41D). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 41G), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 41F). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 41G), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished by flow through deposition (e.g., using a temporarily boundlid), dunk coating, spray coating, puddle dispensing, or by anothersuitable method that will attach the primer(s) 18, 20 or 18′, 20′ to theregion 14 or that will attach the primer(s) 19, 21 or 19′, 21′ to theregion 16. Each of these example techniques may utilize a primersolution or mixture, which may include the primer(s) 18, 20 or 18′, 20′,19, 21 or 19′, 21′ water, a buffer, and a catalyst. With any of thegrafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the substrate26.

Still another example method for making the example shown in FIG. 2 isshown in FIG. 42A through FIG. 42H. Again, while the primer sets 12A,12A′ or 12B, 12B′ or 12C, 12C′ or 12D, 12D′ are mentioned throughoutthis description, they are not shown for clarity.

The substrate 26 may be any examples of the substrate disclosed herein.Similar to the example described in FIG. 41A through FIG. 41G, thesubstrate may also be a multi-layered substrate including anun-patterned resin on a support.

As shown in FIG. 42A, in this example, the sacrificial metal layer 98 isapplied on the substrate 26. The sacrificial metal layer 98 may be anyof the example material and may be deposited by any of the examplesdescribed in reference to FIG. 41A.

As shown in FIG. 42B, a resist is applied to the sacrificial metal layer98 and is patterned to define a multi-level or multi-depth depressiontherein. The patterned resin is shown at reference numeral 54′. In thisexample, the resin may be any nanoimprint lithography resin. In anexample, the resin is spin coated and soft baked, and then stamped andcured (e.g., using ultraviolet curing) to define multi-level ormulti-depth depression that includes a deep portion 70 and the shallowportion 72 which is defined, in part, by a step portion 74 of thepatterned resin 54′.

Wet or dry etching may then be used to expose a portion 100 of thesubstrate 26 (e.g., a portion of the un-patterned resin of themulti-layered substrate) underlying the deep portion 70 and a portion102 of the sacrificial metal layer 98 underlying the shallow portion 72.The exposed portions 100 and 102 are shown in FIG. 42C. Wet or dryetching may be performed as described in reference to FIG. 41C.

As shown in FIG. 42D, the first functionalized layer 60 may be depositedon the remaining patterned resin 54′, on the exposed portion 100 of thesubstrate 26, and on the exposed portion 102 of the sacrificial metallayer 98. The first functionalized layer 60 may be any of the examplesand may be deposited using any of the techniques described herein. Insome instances, the first functionalized layer 60 may also be cured.

In FIG. 42E and FIG. 42F, the first functionalized layer 60 is thenpatterned to form a first functionalized region (region 14) covered by aphotoresist 62. In this example, the photoresist 62 is a negativephotoresist. The photoresist 62 may be applied to the firstfunctionalized layer 60, selectively exposed to certain wavelengths oflight to form an insoluble region, and exposed to a developer solutionto remove the soluble portions. The remaining photoresist 62 ispositioned on the portion of the first functionalized layer 60 that ison the portion 100 in the deep portion 70.

As shown in FIG. 42F, the exposed portions of the first functionalizedlayer 60 (e.g., those not covered by the photoresist 62) may then beremoved, e.g., via etching or another suitable technique. This etchingprocess (e.g., oxygen plasma) also removes some of the patterned resin54′, and some of the photoresist 62. In a separate etch process, thesacrificial metal layer 98 that had been underlying the shallow portion72 (see FIG. 42B) is removed. In this example, wet or dry etching asdescribed in reference to FIG. 41C may be used. This process exposes theother portion 104 of the substrate 26.

As shown in FIG. 42G, the second functionalized layer 64 is thenapplied, using any suitable deposition technique, on the exposedportions of the resin 54′, the photoresist 62, and the exposed portion104 of the substrate 26. In an example, the second functionalized layer64 may be deposited on the photoresist 62, but may be removed with thephotoresist 62 when it is lifted off.

As shown in FIG. 42H, the remaining patterned resin 54′ and thephotoresist 62 may then be lifted off, which removes any of the secondfunctionalized layer 64 thereon. This lift off process may be performedin dimethylsulfoxide (DMSO) using sonication, or in acetone, or with anNMP (N-methyl-2-pyrrolidone) based stripper. The remaining sacrificialmetal layer 98 is then exposed, and can be removed using wet etching asdescribed herein. The regions 14, 16 remain intact on the substratesurface after wet etching, in part because the sacrificial metal layer98 is not present under the regions 14, 16.

In some examples, the primers 18, 20 or 18′, 20′ (not shown in FIG. 42Athrough FIG. 42H) may be pre-grafted to the first functionalized layer60, and thus are attached to the first functionalized region 14.Similarly, the primers 19, 21 or 19′, 21′ (not shown in FIG. 42A throughFIG. 42H) may be pre-grafted to the second functionalized layer 64, andthus are attached to the second functionalized region 16. In theseexamples, additional primer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 42D). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 42H), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 42G). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 42H), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished by flow through deposition (e.g., using a temporarily boundlid), dunk coating, spray coating, puddle dispensing, or by anothersuitable method that will attach the primer(s) 18, 20 or 18′, 20′ to theregion 14 or that will attach the primer(s) 19, 21 or 19′, 21′ to theregion 16. Each of these example techniques may utilize a primersolution or mixture, which may include the primer(s) 18, 20 or 18′, 20′,19, 21 or 19′, 21′ water, a buffer, and a catalyst. With any of thegrafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the substrate26.

The example regions 14, 16 shown in FIG. 2 may also be formed usingmicro-contact printing or direct printing techniques, such as inkjetprinting. These methods may be particularly suitable when it isdesirable to generate regions on a micron scale, such as about 1 μm toabout 50 μm.

In the example shown in FIG. 2, it is to be understood that multiplesets of isolated regions 14, 16 may be formed in an array across thesubstrate surface S. Many different layouts may be used for the array,as long as the regions 14, 16 within an isolated set are adjacent to oneanother.

FIG. 3A through FIG. 6B illustrate different examples where at least oneof the regions 14, 16 is located in a depression 28 defined in thesubstrate 26. The depressions 28 may be formed in a single layeredsubstrate (e.g., substrate 26) or may be formed in an outermost layer ofa multi-layered substrate.

Depressions 28 may be formed using any suitable technique, such asphotolithography, nanoimprint lithography (NIL), stamping techniques,embossing techniques, molding techniques, microetching techniques, etc.

While a single depression 28 is shown in each of FIG. 3A through FIG.6B, it is to be understood that a flow cell may include a plurality ofdepressions 28 that are separated by interstitial regions 30, where eachof the depressions 28 includes the first region 14 located at a firstportion and the second region 16 located at a second portion. Stillfurther, some of the examples disclosed herein include depressions 28Aand 28B with different sizes (see, e.g., FIG. 11C), or depressions 28Cthat include two portions 34, 34′ that are interconnected, but whichhave different sizes (see, e.g., FIG. 11D). It is to be understood thatthe following discussion related to the depressions 28 may be applicablefor any example of the depressions 28, 28A, 28B, 28C disclosed herein.

Many different layouts of the depressions 28 may be envisaged, includingregular, repeating, and non-regular patterns. In an example, thedepressions 28 are disposed in a hexagonal grid for close packing andimproved density. Other layouts may include, for example, rectilinear(i.e., rectangular) layouts (see FIG. 24), triangular layouts, and soforth. In some examples, the layout or pattern can be an x-y format ofdepressions 28 that are in rows and columns. In some other examples, thelayout or pattern can be a repeating arrangement of depressions 28and/or interstitial regions 30. In still other examples, the layout orpattern can be a random arrangement of depressions 28 and/orinterstitial regions 30. The pattern may include stripes, swirls, lines,triangles, rectangles, circles, arcs, checks, plaids, diagonals, arrows,squares, and/or cross-hatches.

The layout or pattern may be characterized with respect to the densityof the depressions 28 (i.e., number of depressions 28) in a definedarea. For example, the depressions 28 may be present at a density ofapproximately 2 million per mm². The density may be tuned to differentdensities including, for example, a density of at least about 100 permm², at least about 1,000 per mm², at least about 0.1 million per mm²,at least about 1 million per mm², at least about 2 million per mm², atleast about 5 million per mm², at least about 10 million per mm², atleast about 50 million per mm², or more. Alternatively or additionally,the density may be tuned to be no more than about 50 million per mm², nomore than about 10 million per mm², no more than about 5 million permm², no more than about 2 million per mm², no more than about 1 millionper mm², no more than about 0.1 million per mm², no more than about1,000 per mm², no more than about 100 per mm², or less. It is to befurther understood that the density of depressions 28 can be between oneof the lower values and one of the upper values selected from the rangesabove. As examples, a high density array may be characterized as havingdepressions 28 separated by less than about 100 nm, a medium densityarray may be characterized as having depressions 28 separated by about400 nm to about 1 μm (1000 nm), and a low density array may becharacterized as having depressions 28 separated by greater than about 1μm. While example densities have been provided, it is to be understoodthat substrates with any suitable densities may be used.

The layout or pattern of the depressions 28 may also or alternatively becharacterized in terms of the average pitch, i.e., the spacing from thecenter of the depression 28 to the center of an adjacent depression 28(center-to-center spacing) or from the edge of one depression 28 to theedge of an adjacent depression 28 (edge-to-edge spacing). The patterncan be regular, such that the coefficient of variation around theaverage pitch is small, or the pattern can be non-regular in which casethe coefficient of variation can be relatively large. In either case,the average pitch can be, for example, at least about 10 nm, about 0.1μm, about 0.5 μm, about 1 μm, about 5 μm, about 10 μm, about 100 μm, ormore. Alternatively or additionally, the average pitch can be, forexample, at most about 100 μm, about 10 μm, about 5 μm, about 1 μm,about 0.5 μm, about 0.1 μm, or less. The average pitch for a particularpattern of depressions 28 can be between one of the lower values and oneof the upper values selected from the ranges above. In an example, thedepressions 28 have a pitch (center-to-center spacing) of about 1.5 μm.While example average pitch values have been provided, it is to beunderstood that other average pitch values may be used.

In the example shown in FIG. 3A through FIG. 6B, the depressions 28 arewells. The wells may be micro wells or nanowells. The size of each wellmay be characterized by its volume, well opening area, depth, and/ordiameter.

Each well can have any volume that is capable of confining a liquid. Theminimum or maximum volume can be selected, for example, to accommodatethe throughput (e.g., multiplexity), resolution, analyte composition, oranalyte reactivity expected for downstream uses of the flow cell. Forexample, the volume can be at least about 1×10⁻³ μm³, at least about1×10⁻² μm³, at least about 0.1 μm³, at least about 1 μm³, at least about10 μm³, at least about 100 μm³, or more. Alternatively or additionally,the volume can be at most about 1×10⁴ μm³, at most about 1×10³ μm³, atmost about 100 μm³, at most about 10 μm³, at most about 1 μm³, at mostabout 0.1 μm³, or less. It is to be understood that the region(s) 14, 16can fill all or part of the volume of a well. The volume of, forexample, the polymer layer in an individual well can be greater than,less than or between the values specified above.

The area occupied by each well opening on a surface can be selectedbased upon similar criteria as those set forth above for well volume.For example, the area for each well opening on a surface can be at leastabout 1×10⁻³ μm², at least about 1×10⁻² μm², at least about 0.1 μm², atleast about 1 μm², at least about 10 μm², at least about 100 μm², ormore. Alternatively or additionally, the area can be at most about 1×10³μm², at most about 100 μm², at most about 10 μm², at most about 1 μm²,at most about 0.1 μm², about 1×10⁻² μm², or less. The area occupied byeach well opening can be greater than, less than or between the valuesspecified above.

The depth of each well (or any other type of depression 28) can be atleast about 0.1 μm, at least about 1 μm, at least about 10 μm, at leastabout 100 μm, or more. Alternatively or additionally, the depth can beat most about 1 ×10³ μm, at most about 100 μm, at most about 10 μm, atmost about 1 μm, at most about 0.1 μm, or less. The depth of each well(or other depression 28) can be greater than, less than or between thevalues specified above.

In some instances, the diameter of each well (or other depression 28)can be at least about 50 nm, at least about 0.1 μm, at least about 0.5μm, at least about 1 μm, at least about 10 μm, at least about 100 μm, ormore. Alternatively or additionally, the diameter can be at most about1×10³ μm, at most about 100 μm, at most about 10 μm, at most about 1 μm,at most about 0.5 μm, at most about 0.1 μm, or less (e.g., about 50 nm).The diameter of each well (or other depression 28) can be greater than,less than or between the values specified above.

When the depression 28 is a trench (see, e.g., FIG. 34H), both thetrenches and interstitial regions can have a rectilinear configuration.The depth of each trench can be at least at least about 0.02 μm (20 nm),at least about 0.1 μm (100 nm), at least about 1 μm, at least about 10μm, at least about 100 μm, or more. Alternatively or additionally, thedepth can be at most about 1 ×10³ μm, at most about 100 μm, at mostabout 10 μm, at most about 1 μm, at most about 0.1 μm, or less. Thedepth of each trench can be greater than, less than or between thevalues specified above.

In some instances, the width of each trench can be at least about 0.02μm, at least about 0.1 μm, at least about 0.5 μm, at least about 1 μm,at least about 10 μm, at least about 100 μm, or more. Alternatively oradditionally, the width can be at most about 1 ×10³ μm, at most about100 μm, at most about 10 μm, at most about 1 μm, at most about 0.5 μm,at most about 0.1 μm, or less (e.g., about 50 nm). The width of eachtrench can be greater than, less than or between the values specifiedabove.

FIG. 3A and FIG. 3B depict, respectively, a cross-sectional view and atop view of an example where the regions 14, 16 are located in differentareas of the depression 28. In this example, the regions 14, 16 aredirectly adjacent to each other within the depression 28.

One example method for making the example shown in FIG. 3A and FIG. 3Bis shown in FIG. 28A through FIG. 28G. While the primer sets 12A, 12A′or 12B, 12B′ or 12C, 12C′ or 12D, 12D′ are mentioned throughout thisdescription, they are not shown for clarity. Moreover, FIG. 3A and FIG.3B depict the depression 28 defined in a single layer substrate 26,while the example method depicts the depression 28 defined in apatterned resin 54′ on a support 52 of a multi-layered substrate. It isto be understood that this method may be used with a single layersubstrate.

In this example, the multi-layered substrate includes a (patterned)resin 54′ on a support 52. Any example of the resin 54 (e.g., see thesection “Bead Based Flow Cell”), the support 52, and the methods forpatterning the resin 54 described herein may be used.

As shown in FIG. 28A, the depression 28 defined in the patterned resin54′ is adjacent to interstitial regions 30, which separate adjacentdepressions 28 from one another. As shown in FIG. 28B, a firstfunctionalized layer 60 is applied (e.g., deposited or deposited andcured) on the patterned resin 54′ using any of the techniques describedherein.

In FIG. 28C and FIG. 28D, the first functionalized layer 60 is thenpatterned to form a first functionalized region (region 14) covered by aphotoresist 62. In this example, the photoresist 62 may be a negativephotoresist (exposed region becomes insoluble) or a positive photoresist(exposed region becomes soluble). The photoresist 62 is applied to thefirst functionalized layer 60, is selectively exposed to certainwavelengths of light to form the insoluble or soluble region, and isexposed to a developer solution to remove the soluble portions. In otherexamples, the photoresist 62 may be replaced with a nanoimprintlithography resin. As shown in FIG. 28C, in this example, thephotoresist 62 covers a portion of the first functionalized layer 60that is on a first portion 76 of the depression 28 (e.g., the portion ofthe layer 60 that is to become the region 14) and does not cover asecond portion of the first functionalized layer 60 that is on a secondportion 78 of the depression 28.

As shown in FIG. 28D, the exposed portions of the first functionalizedlayer 60 (e.g., those not covered by the photoresist 62) may then beremoved, e.g., via etching or another suitable technique. This exposesthe second portion 78 of the depression 28 and the interstitial regions30.

As shown in FIG. 28E, the second functionalized layer 64 is thenapplied, using any suitable deposition technique (with or without curingdepending upon the material), on the photoresist 62 and on portions(e.g., the exposed surface S) of the substrate 26, including on thesecond portion 78 of the depression 28 and on the interstitial regions30.

As shown in FIG. 28F, the photoresist 62 may then be lifted off, whichalso removes any of the second functionalized layer 64 thereon.

In FIG. 28G, a portion of the second functionalized layer 64 is removed.In particular, the second functionalized layer 64 is removed from theinterstitial regions 30. In this example, removing involves polishingthe second functionalized layer 64 (and any of the first functionalizedlayer 60 that may be present) from the interstitial regions 30.

The polishing process may be performed with a gentle chemical slurry(including, e.g., an abrasive, a buffer, a chelating agent, asurfactant, and/or a dispersant) which can remove the secondfunctionalized layer 64 (and any of the first functionalized layer 60that may be present) from the interstitial regions 30 withoutdeleteriously affecting the underlying substrate 26 or patterned resin54′ at those regions. Alternatively, polishing may be performed with asolution that does not include the abrasive particles.

The chemical slurry may be used in a chemical mechanical polishingsystem to polish the surface of the interstitial regions 30. Thepolishing head(s)/pad(s) or other polishing tool(s) is/are capable ofpolishing the second functionalized layer 64 (and any of the firstfunctionalized layer 60 that may be present) from the interstitialregions 30 while leaving the regions 14, 16 in the depression(s) 28 atleast substantially intact. As an example, the polishing head may be aStrasbaugh ViPRR II polishing head.

Cleaning and drying processes may be performed after polishing. Thecleaning process may utilize a water bath and sonication. The water bathmay be maintained at a relatively low temperature ranging from about 22°C. to about 30° C. The drying process may involve spin drying, or dryingvia another suitable technique.

In some examples, the primers 18, 20 or 18′, 20′ (not shown in FIG. 28Athrough FIG. 28G) may be pre-grafted to the first functionalized layer60, and thus are attached to the first functionalized region 14.Similarly, the primers 19, 21 or 19′, 21′ (not shown in FIG. 28A throughFIG. 28G) may be pre-grafted to the second functionalized layer 64, andthus are attached to the second functionalized region 16. In theseexamples, additional primer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 28B). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 28G), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 28E). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 28G), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished using any grafting technique disclosed herein. With any ofthe grafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the patternedresin 54′.

While not shown in FIG. 28A through FIG. 28G, this example method mayfurther include depositing a first self-assembled monolayer (SAM) on thefirst functionalized region 14 and depositing a second self-assembledmonolayer (SAM) on the second functionalized region 16. In the exampleusing the self-assembled monolayers, the primer sets 12A, 12A′ or 12B,12B′ or 12C, 12C′ or 12D, 12D′ are grafted after the SAMS are formed.

Another example method for making the example shown in FIG. 3A and FIG.3B is shown in FIG. 29A through FIG. 29H. While the primer sets 12A,12A′ or 12B, 12B′ or 12C, 12C′ or 12D, 12D′ are mentioned throughoutthis description, they are not shown for clarity. Moreover, FIG. 3A andFIG. 3B depict the depression 28 defined in a single layer substrate 26,while the example method depicts the depression 28′ defined in apatterned resin 54′ on a support 52 of a multi-layered substrate. It isto be understood that this method may be used with a single layersubstrate.

In this example, the multi-layered substrate includes the (patterned)resin 54′ on the support 52. As shown in FIG. 29A, the depression 28′defined in the patterned resin 54′ is adjacent to interstitial regions30, which separate adjacent depressions 28′ from one another. Thedepression 28′ is a multi-level or multi-depth depression that includesa deep portion 70 and a shallow portion 72 which is defined, in part, bya step portion 74 of the patterned resin 54′.

In this example, a sacrificial layer is applied on the patterned resin54′ so that the sacrificial layer at least partially fills the deepportion 70 in the depression 28′. An example sacrificial layer 76 is anymaterial that has an etch rate that is different than the resin 54′ anda photoresist 62 used in the method. Examples of suitable sacrificiallayer materials 76 include silicon, aluminum, negative or positivephotoresists, copper, etc. These materials may be deposited using anysuitable technique disclosed herein. While not shown, it is to beunderstood that in addition to being deposited in at least part of thedeep portion 70, the sacrificial layer may also be deposited on theinterstitial regions 30 and on the step portion 74, or to completelyfill the depression 28′.

A portion of the sacrificial layer and a portion of the resin 54′ arethen sequentially removed. The sacrificial layer may first be etchedback so that it is removed from the interstitial regions 30 and from thestep portion 74, and so that the remaining portion of the sacrificiallayer (shown at reference numeral 80 in FIG. 29B) in the deep portion 70is substantially level with the step portion 74. As shown in FIG. 29B,several portions of the resin 54′ are removed. For example, portions ofthe resin 54′ are removed to form new (referred to as second)interstitial regions 30′ that are substantially level with the remainingportion of the sacrificial layer 80, and the resin 54′ is removed to getrid of the step portion 74. Removal of the step portion 74 forms anarea/portion 76 of the depression 28′ next to the remaining portion ofthe sacrificial layer 80.

As shown in FIG. 29C, the first functionalized layer 60 is applied onthe remaining portion of the sacrificial layer 80, the area/portion 76,and the (second) interstitial regions 30′. As shown in FIG. 29D, aphotoresist 62 is then applied on the first functionalized layer 60.

Portions of the photoresist 62 and the underlying first functionalizedlayer 60 may then be removed to expose the remaining portion of thesacrificial layer 80 and the interstitial regions 30′. This is shown inFIG. 29E. This removal process may be accomplished by etching with anetchant that selectively removes the portion of the photoresist 62 andthe underlying first functionalized layer 60, but does not remove theremaining portion of the sacrificial layer 80. In this example, wetetching may be performed with a basic pH developer solution, such asNaOH, KOH, or TMAH (tetramethylammonium hydroxide), or dry etching maybe performed with an oxygen plasma. In this example, etching is stoppedwhen the remaining portion of the sacrificial layer 80 is exposed. Thisleaves a second portion (e.g., region 14) of the first functionalizedlayer 60 having a second portion 62″ of the photoresist thereon at thearea/portion 76.

As shown at FIG. 29F, the remaining portion of the sacrificial layer 80is then removed to expose a second area/portion 78 next to the secondportion (e.g., region 14) of the first functionalized layer 60. Thisremoval process may be accomplished by etching with an etchant thatselectively removes the remaining sacrificial layer 76, but does notremove second portion (e.g., region 14) of the first functionalizedlayer 60 having a second portion 62″ of the photoresist thereon. Asexamples, an aluminum sacrificial layer 80 can be removed in acidic orbasic conditions, a copper sacrificial layer 80 can be removed usingFeCl₃, a photoresist sacrificial layer 80 can be removed using organicsolvents, such as acetone, or in basic (pH) conditions; and a siliconsacrificial layer 80 can be removed in basic (pH) conditions.

As shown in FIG. 29G, the second functionalized layer 64 is then appliedto the area/portion 78, using any suitable deposition (and ifapplicable, curing) technique. This forms the second functionalizedregion 16. As shown in FIG. 29G, the second functionalized layer 64 mayalso be applied on the second portion 62″ of the photoresist and on theinterstitial regions 30.

As shown in FIG. 29G, the second portion 62″ of the photoresist may thenbe lifted off, which also removes any of the second functionalized layer64 thereon. This forms the first functionalized region 14. Polishing mayalso be performed to remove the second functionalized layer 64 from theinterstitial regions 30′.

While the example shown in FIG. 29A through 29H includes the photoresist62, it is to be understood that if the second functionalized layer 64does not adhere to the first functionalized layer 60, then thephotoresist 62 may be omitted. Moreover, if the sacrificial layer 80 isa transparent material that the first functionalized layer 60 does notadhere to, then the sacrificial layer 80 may not be removed and theregion 16 may be formed on the sacrificial layer 80.

In some examples, the primers 18, 20 or 18′, 20′ (not shown in FIG. 29Athrough FIG. 29H) may be pre-grafted to the first functionalized layer60, and thus are attached to the first functionalized region 14.Similarly, the primers 19, 21 or 19′, 21′ (not shown in FIG. 29A throughFIG. 29H) may be pre-grafted to the second functionalized layer 64, andthus are attached to the second functionalized region 16. In theseexamples, additional primer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 29C). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 29H), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 29G). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 29H), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished using any grafting technique disclosed herein. With any ofthe grafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the patternedresin 54′.

While not shown in FIG. 29A through FIG. 29H, this example method mayfurther include depositing a first self-assembled monolayer (SAM) on thefirst functionalized region 14 and depositing a second self-assembledmonolayer (SAM) on the second functionalized region 16. In the exampleusing the self-assembled monolayers, the primer sets 12A, 12A′ or 12B,12B′ or 12C, 12C′ or 12D, 12D′ are grafted after the SAMS are formed.

Still another example method for making the example shown in FIG. 3A andFIG. 3B is shown in FIG. 30A through FIG. 30F. While the primer sets12A, 12A′ or 12B, 12B′ or 12C, 12C′ or 12D, 12D′ are mentionedthroughout this description, they are not shown for clarity. Moreover,FIG. 3A and FIG. 3B depict the depression 28′ defined in a single layersubstrate 26, while the example method depicts the depression 28′defined in a patterned resin 54′ on a support 52 of a multi-layeredsubstrate. It is to be understood that this method may be used with asingle layer substrate.

As shown in FIG. 30A, in this example, the multi-layered substrateincludes an un-patterned resin 54 on the support 52, and the sacrificiallayer 80 on the resin 54. Any examples of the resin 54, support 52, andsacrificial layer 80 may be used.

As shown in FIG. 30B, additional resin is applied to the sacrificiallayer 80 and is patterned to define the depression 28′ therein. Theadditional resin may be the same as or different than the resin 54. Thedepression 28′ is a multi-level or multi-depth depression that includesthe deep portion 70 and the shallow portion 72 which is defined, inpart, by a step portion 74 of the patterned resin 54′.

As shown in FIG. 30C, a first portion of the patterned resin 54′(adjacent to the deep portion 70) and a portion of the sacrificial layer80 underlying the deep portion 70 are etched. This exposes a portion 82of the resin 54. In an example, the patterned resin 54′ may be etchedusing an anisotropic oxygen plasma to expose the underlying portion ofthe sacrificial layer 80, and then the portion of the sacrificial layer80 may be removed, e.g., using a BCl₃ and Cl₂ plasma.

As shown in FIG. 30D, the step portion 74 of the patterned resin 54′ isetched away, e.g., using oxygen plasma. This exposes the sacrificiallayer 80 that underlies the step portion 74. The etchant used can etchthe resin 54′, but not the sacrificial layer 80. As such, thesacrificial layer 80 acts as an etch stop, and thus portion 102′ of thesacrificial layer 80 is exposed.

It is to be understood that when the resin 54′ is etched, the initialinterstitial regions 30 may be shortened. As such, the interstitialregions 30′ are formed.

Also as shown in FIG. 30D, the first functionalized layer 60 may bedeposited (and cured in some instances). The first functionalized layer60 may not adhere to the exposed portion 102′ of the sacrificial layer80, but will adhere to the portion 82 of the resin 54 and to theinterstitial regions 30′ surrounding the depression 28′.

The exposed portion 102′ of the sacrificial layer 80 may then be etchedaway (e.g., using a basic solution or FeCl₃ depending upon thematerial), and the second functionalized layer 64 may be deposited (andcured depending upon the material). As shown in FIG. 30E, the secondfunctionalized layer 64 is applied to the newly exposed portion of theresin 54 (where portion 102′ has been removed). In one example, thefirst functionalized layer 60 has no affinity for the secondfunctionalized layer 64, and thus the second functionalized layer 64does not deposit on the first functionalized layer 60. In this exampleof the method, as shown in FIG. 30F, polishing may be performed toremove the first functionalized layer 60 from the interstitial regions30′.

In some examples of the method(s) of FIG. 30A through FIG. 30F, theprimers 18, 20 or 18′, 20′ (not shown in FIG. 30A through FIG. 30F) maybe pre-grafted to the first functionalized layer 60, and thus areattached to the first functionalized region 14. Similarly, the primers19, 21 or 19′, 21′ (not shown in FIG. 30A-FIG. 30) may be pre-grafted tothe second functionalized layer 64, and thus are attached to the secondfunctionalized region 16. In these examples, additional primer graftingis not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 30A or 30D). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 30F), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 30E). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 30F), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished using any grafting technique disclosed herein. With any ofthe grafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the patternedresin 54′.

While not shown in FIG. 30A through FIG. 30F, this example method mayfurther include depositing a first self-assembled monolayer (SAM) on thefirst functionalized region 14 and depositing a second self-assembledmonolayer (SAM) on the second functionalized region 16. In the exampleusing the self-assembled monolayers, the primer sets 12A, 12A′ or 12B,12B′ or 12C, 12C′ or 12D, 12D′ are grafted after the SAMS are formed.

Still another example method for making the example shown in FIG. 3A andFIG. 3B is shown in FIG. 31A through FIG. 31I. While the primer sets12A, 12A′ or 12B, 12B′ or 12C, 12C′ or 12D, 12D′ are mentionedthroughout this description, they are not shown for clarity. Moreover,FIG. 3A and FIG. 3B depict the depression 28′ defined in a single layersubstrate 26, while the example method depicts the depression 28′defined in a patterned resin 54′ on a support 52 of a multi-layeredsubstrate. It is to be understood that this method may be used with asingle layer substrate.

As shown at FIG. 31A, the multi-layered substrate includes the(patterned) resin 54′ on the support 52. The depression 28′ defined inthe patterned resin 54′ is adjacent to interstitial regions 30, whichseparate adjacent depressions 28′ from one another. The depression 28′is a multi-level or multi-depth depression that includes a deep portion70 and a shallow portion 72 which is defined, in part, by a step portion74 of the patterned resin 54′.

As shown in FIG. 31B, a first functionalized layer 60 is applied on thepatterned resin 54′. The first functionalized layer 60 may be any of theexamples disclosed herein and may be deposited using any of thetechniques described herein.

In FIG. 31C and FIG. 31D, the first functionalized layer 60 is thenpatterned to form a first functionalized region (region 14) covered by aphotoresist 62. In this example, the photoresist 62 is a negativephotoresist (exposed region becomes insoluble). As shown in FIG. 31C,the photoresist 62 is applied to the first functionalized layer 60, isselectively exposed to certain wavelengths of light to form theinsoluble region (shown at 62 in FIG. 31D), and is exposed to adeveloper solution to remove the soluble portions. As shown in FIG. 31D,the exposed portions of the first functionalized layer 60 (e.g., thosenot covered by the photoresist 62) may then be removed, e.g., viaetching or another suitable technique. The remaining portion of thefirst functionalized layer 60 (e.g., the region 14) is at a first levelof each multi-level depression. In this example, the first level is indeep portion 70 on the support 52.

In this example, a sacrificial layer 84 is applied on the photoresist 62and portions of the resin 54′ (e.g., interstitial regions 30, surface ofstep portion 74). The sacrificial layer 84 is shown in FIG. 31E. Anymaterial may be used as the sacrificial layer 84 that has an etchdifferential relative to the resin 54′. In an example, aluminum may beused as the sacrificial layer 84.

As shown in FIG. 31F, the sacrificial layer 84 is removed from theportions of the resin 54′. The sacrificial layer 84 may first be etchedback so that it is removed from the interstitial regions 30 and from thestep portion 74, and so that the remaining portion of the sacrificiallayer (shown at reference numeral 84′ in FIG. 31F) remains on thephotoresist 62 in the deep portion 70.

A region of the resin 54′ (specifically the step region 74) is thenremoved from the multi-layer depression 28′ to create an area/portion 78that is adjacent to the first functionalized region 14 (e.g., theportion of the first functionalized layer 60 that underlies thephotoresist 62 and the remaining portion of the sacrificial layer 84′.This process may also remove portions of the interstitial regions 30,resulting in new interstitial regions 30′. This removal process may beaccomplished by etching with an etchant, such as oxygen plasma, thatselectively removes the resin 54′, but does not remove the remainingportion of the sacrificial layer 84′.

As shown in FIG. 31H, the second functionalized layer 64 is thenapplied, using any suitable deposition technique, on the remainingportion of the sacrificial layer 84, on the area/portion 78, and on theinterstitial regions 30′.

As shown in FIG. 31I, the photoresist 62 may then be lifted off, whichalso removes the remaining portion of the sacrificial layer 84′ and anyof the second functionalized layer 64 thereon. Any of the secondfunctionalized layer 64 on the interstitial regions 30′ may also beremoved via polishing.

In some examples of the method(s) of FIG. 31A through FIG. 31I, theprimers 18, 20 or 18′, 20′ (not shown in FIG. 31A through FIG. 31I) maybe pre-grafted to the first functionalized layer 60, and thus areattached to the first functionalized region 14. Similarly, the primers19, 21 or 19′, 21′ (not shown in FIG. 31A through FIG. 31I) may bepre-grafted to the second functionalized layer 64, and thus are attachedto the second functionalized region 16. In these examples, additionalprimer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 31B). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 31I), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 31H). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 31I), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished using any grafting technique disclosed herein. With any ofthe grafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the patternedresin 54′.

While not shown in FIG. 31A through FIG. 31I, this example method mayfurther include depositing a first self-assembled monolayer (SAM) on thefirst functionalized region 14 and depositing a second self-assembledmonolayer (SAM) on the second functionalized region 16. In the exampleusing the self-assembled monolayers, the primer sets 12A, 12A′ or 12B,12B′ or 12C, 12C′ or 12D, 12D′ are grafted after the SAMS are formed.

Throughout the processing in the examples shown in FIG. 31A through FIG.31I, the multi-level depression 28′ becomes a single-level depression28, as shown in FIG. 3A.

FIGS. 4A through 4C illustrate different examples of the regions 14, 16that are located in different portions of the depression 28.

In FIG. 4A, there is a gap 36 between the regions 14, 16. As such, thisexample of the flow cell includes a gap 36 separating the first primerset 12A, 12B, 12C, 12D (at region 14, not shown in FIG. 4A) and thesecond primer set 12A′, 12B′, 12C′, 12D′ (at region 16, not shown inFIG. 4B). In one example, the gap 36 is a space between respectivepolymer sections where the regions 14, 16 are formed. Any of the methodmethods shown in FIG. 31A through FIG. 31I may be modified to form thegap 36. The gap 36 may have any measurable length greater than zero. Inan example, the gap 36 is greater is 1 nm. In an example, the gap rangesfrom about 1 nm to about 10 nm.

In FIG. 4B, the regions 14, 16 partially overlap. The overlapping region38 is an area where both primers 18, 20 or 18′, 20′ and primers 19, 21or 19′, 21′ are grafted. In an example, this overlapping region 38 maybe formed during the patterning and grafting process by having the sameportion of the polymer layer 32 exposed when the primers 18, 20 or 18′,20′ are grafted and when the primers 19, 21 or 19′, 21′ are grafted. Inanother example, this overlapping region 38 may be formed whenseparately grafted primers 18, 20 or 18′, 20′ and primers 19, 21 or 19′,21′ physically overlap or interdiffuse during or after the process.

In FIG. 4C, the first and second portions of the depression 28′ wherethe respective regions 14, 16 are generated have different depths. Anyof the depression depths described herein may be used, as long as oneportion of the depression 28′ is deeper than the other portion of thedepression 28′. The different depths may be generated when thedepression 28′ is formed, e.g., via nanoimprinting, etching, etc.

One example method for making the example shown in FIG. 4C is shown inFIG. 32A through FIG. 32F. While the primer sets 12A, 12A′ or 12B, 12B′or 12C, 12C′ or 12D, 12D′ are mentioned throughout this description,they are not shown for clarity. Moreover, FIG. 4C depicts the depression28′ defined in a single layer substrate 26, while the example methoddepicts the depression 28′ defined in a patterned resin 54′ on a support52 of a multi-layered substrate. It is to be understood that this methodmay be used with a single layer substrate.

As shown at FIG. 32A, the multi-layered substrate includes the(patterned) resin 54′ on the support 52. The depression 28′ defined inthe patterned resin 54′ is adjacent to interstitial regions 30, whichseparate adjacent depressions 28′ from one another. The depression 28′is a multi-level or multi-depth depression that includes a deep portion70 and a shallow portion 72 which is defined, in part, by a step portion74 of the patterned resin 54′.

As shown in FIG. 32B, a first functionalized layer 60 is applied on thepatterned resin 54′. The first functionalized layer 60 may be any of theexamples disclosed herein and may be deposited using any of thetechniques described herein, and if suitable, may also be cured.

In FIG. 32C and FIG. 32D, the first functionalized layer 60 is thenpatterned to form a first functionalized region (region 14) covered by aphotoresist 62. In this example, the photoresist 62 is a negativephotoresist or a positive photoresist. As shown in FIG. 32C, thephotoresist 62 is applied to the first functionalized layer 60, isselectively exposed to certain wavelengths of light to form theinsoluble region or the soluble region (depending on the resist used),and is exposed to a developer solution to remove the soluble portions.As shown in FIG. 32D, the exposed portions of the first functionalizedlayer 60 (e.g., those not covered by the photoresist 62) may then beremoved, e.g., via etching or another suitable technique. In thisexample, the first functionalized layer 60 and the resin 54′ may have anetch differential. The remaining portion of the first functionalizedlayer 60 (e.g., region 14) is at a first level of each multi-leveldepression. In this example, the first level is in deep portion 70 onthe resin 54′.

As shown in FIG. 32E, the second functionalized layer 64 is thenapplied, using any suitable deposition technique, on the exposedportions of the resin 54′ (e.g., on the interstitial regions 30 and onthe step region 74). Depending on the material, the secondfunctionalized layer 64 may also be cured. In one example, thephotoresist 62 has no affinity for the second functionalized layer 64,and thus the second functionalized layer 64 does not deposit on thephotoresist 62. In another example, the second functionalized layer 64may be deposited on the photoresist 62, but may be removed with thephotoresist 62 is lifted off.

As shown in FIG. 32F, the photoresist 62 may then be lifted off, which,in some instances, also removes any of the second functionalized layer64 thereon. Any of the second functionalized layer 64 on theinterstitial regions 30 may also be removed via polishing.

In some examples of the method(s) of FIG. 32A through FIG. 32F, theprimers 18, 20 or 18′, 20′ (not shown in FIG. 32A through FIG. 32F) maybe pre-grafted to the first functionalized layer 60, and thus areattached to the first functionalized region 14. Similarly, the primers19, 21 or 19′, 21′ (not shown in FIG. 32A through FIG. 32F) may bepre-grafted to the second functionalized layer 64, and thus are attachedto the second functionalized region 16. In these examples, additionalprimer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 32B). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 32F), because they will not graft tothe surface functional groups of the region 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 32E). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 32F), because they will not graft tothe surface functional groups of the region 14.

When grafting is performed during the method, grafting may beaccomplished using any grafting technique disclosed herein. With any ofthe grafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for the patternedresin 54′.

While not shown in FIG. 32A through FIG. 32F, this example method mayfurther include depositing a first self-assembled monolayer (SAM) on thefirst functionalized region 14 and depositing a second self-assembledmonolayer (SAM) on the second functionalized region 16. In the exampleusing the self-assembled monolayers, the primer sets 12A, 12A′ or 12B,12B′ or 12C, 12C′ or 12D, 12D′ are grafted after the SAMS are formed.

FIGS. 5, 6A and 6B illustrate different examples where the substrate 26includes depressions 28 separated by interstitial regions 30; each ofthe depressions includes the first region 14; and the second region 16is located on at least some of the interstitial regions 30. As such, inthese examples, one of the regions 14 is located in the depression 28and the other of the regions 16 is located on the substrate surface Sadjacent to the depression 28. In the example shown in FIG. 5, theregion 16 on the substrate surface S is next to the depression 28. Inthe example shown in FIGS. 6A and 6B, the region 16 on the substratesurface S surrounds the depression 28. Example methods for making one ofthe regions 14, 16 in the depression 28 and the other of the regions 16,14 on at least a portion of the substrate surface S will now bedescribed in reference to FIG. 7A through FIG. 7G and FIG. 8A throughFIG. 8F.

One example method for making the example shown in FIGS. 6A and 6B isshown in FIGS. 7A through 7G. As shown in FIG. 7A, the method utilizesthe substrate 26 having a plurality of depressions 28 separated byinterstitial regions 30. In this example method, the polymer layer 32 isdeposited on the substrate 26 and polished from the interstitial regions30 as described in reference to FIGS. 3A and 3B. This leaves the polymerlayer 32 in the depression 28 and not on the interstitial regions 30. Ifit is desirable for the regions 14 to be in the depressions 28, then theprimers 18, 20 or 18′, 20′ may be grafted using any of the examplesdisclosed herein. If it is desirable for the regions 16 to be in thedepressions 28, then the primers 19, 21 or 19′, 21′ may be grafted usingany of the examples disclosed herein. In the example shown in FIG. 7B,the primers 18, 20 or 18′, 20′ are grafted to the polymer layer 32. Itis to be understood that the primers 18, 20 or 18′, 20′ will graft tothe polymer layer 32 in the depression 28 and will not graft to theinterstitial regions 30, as shown in FIG. 7B.

In some examples of this method, the substrate 26 (which has the region14 or 16 formed in the depression 28) is exposed to a capping agent. Thecapping agent includes a chemical species that can react with anyunreacted functional groups of the polymer layer 32 (e.g., anyfunctional groups that have not reacted with a primer 18, 20 or 18′,20″) in order to render these functional groups non-functional duringsubsequent processing. This process may reduce the ability of thesubsequently deposited polymer layer 32′ to adhere to the region 14. Inother words, the interaction between the polymer layer 32 and thesubsequently deposited polymer layer 32′ is reduced so that little or nopolymer layer 32′ adheres to the polymer layer 32. This, in turn,reduces the ability of subsequently deposited primers (e.g., 19, 21 or19′, 21′) to graft to the area overlying the region 14.

In an example where the polymer layer 32 includes azide functionalgroups, the chemical species in the capping agent may be a reducingagent, such as a phosphine. An example phosphine istris(hydroxypropyl)phosphine. The non-reacted azides of the polymerlayer 32 will be reduced by the phosphine, which renders themnon-functional for additional primer grafting.

As shown in FIG. 7C, the method then includes depositing the secondpolymer layer 32′. The polymer layer 32′ may be the same polymer that isused in the polymer layer 32, or may be a different type of polymer thanthat used in the polymer layer 32. In an example, each of the firstpolymer 32 and the second polymer 32′ is an acrylamide copolymer, suchas PAZAM. The polymer layer 32′ may coat the interstitial regions 30alone, or may coat at least some of the regions 14 in addition to theinterstitial regions 30. Whether the polymer layer 32′ at leastpartially coats the regions 14 will depend upon the capping agent usedon the polymer layer 32.

In this example, template polynucleotide strands, including anun-cleavable first template strand 40 and a cleavable second templatestrand 42, may be formed in the region 14 using, respectively, theprimers 18 or 18′ and 20, 20′. At the outset of template polynucleotidestrand formation, library templates may be prepared from any nucleicacid sample (e.g., a DNA sample or an RNA sample). The nucleic acidsample may be fragmented into single-stranded, similarly sized (e.g.,<1000 bp) DNA or RNA fragments. During preparation, adapters may beadded to the ends of these fragments. Through reduced cycleamplification, different motifs may be introduced in the adapters, suchas sequencing binding sites, indices, and regions that are complementaryto the primers 18 or 18′ and 20, 20′ in the regions 14. The finallibrary templates include the DNA or RNA fragment and adapters at bothends. In some examples, the fragments from a single nucleic acid samplehave the same adapters added thereto.

A plurality of library templates may be introduced to the substrate 26.This may involve introducing a template fluid to the flow cell. Thetemplate fluid may include a liquid carrier and the plurality of librarytemplates. Because the substrate 26 includes an array of regions 14 inthe depressions 28, multiple library templates are hybridized, forexample, to one of two types of primers 18, 18′ or 20, 20′ immobilizedtherein.

Cluster generation may then be performed. During cluster generation, atemplate from the template fluid is amplified to form a cluster in atleast some of the depressions 28. In one example of cluster generation,the library templates are copied from the hybridized primers 18, 18′ or20, 20′ by 3′ extension using a high-fidelity DNA polymerase. Theoriginal library templates are denatured, leaving the copies immobilizedin the regions 14. Isothermal bridge amplification may be used toamplify the immobilized copies. For example, the copied templates loopover to hybridize to an adjacent, complementary primer 20, 20′ or 18,18′, and a polymerase copies the copied templates to form doublestranded bridges, which are denatured to form two single strandedstrands. These two strands loop over and hybridize to adjacent,complementary primers 20, 20′ or 18, 18′ and are extended again to formtwo new double stranded loops. The process is repeated on each templatecopy by cycles of isothermal denaturation and amplification to createdense clonal clusters. Each cluster of double stranded bridges isdenatured, resulting in un-cleavable first template strands 40 attachedto the un-cleavable first primers 18, 18′ and cleavable second templatestrands 42 attached to the cleavable second primers 20, 20′ as shown inFIG. 7D. It is to be understood that the cleavability of the primersdrives the cleavability of the template strands attached thereto.Because the second template strands 42 attached to the cleavable secondprimers 20, 20′ include the cleavage site 22, the cleavable secondtemplate strands 42 are cleavable. This example of clustering is bridgeamplification, and is one example of the amplification that may beperformed. It is to be understood that other amplification techniquesmay be used, such as the exclusion amplification (Examp) workflow(Illumina Inc.).

It is to be understood that because the second polymer layer 32′ doesnot have primers 18, 18′ or 20, 20′ grafted thereto, the amplificationprocess does not extend beyond the individual depressions 28.

A priming fluid may then be introduced to the substrate 26. The primingfluid includes a liquid carrier and a second primer set 12A′, 12B′,12C′, 12D′ that is different from the first primer set 12A, 12B, 12C,12D that has been introduced into the depressions 28. The liquid carrierin the priming fluid may be any liquid that can support click chemistry,such as phosphate buffered saline (PBS), saline-sodium citrate (SSC), acarbonate based buffer, etc. In this example, the primer set in thepriming fluid includes primers 19 or 19′ and 21 or 21′. It is to beunderstood that if the primer set 12A′, 12B′, 12C′, 12D′ (includingprimers 19 or 19′ and 21 or 21′) is grafted first to form the region 16in the depressions 28, then the priming fluid may include the primer set12A, 12B, 12C, 12D (including primers 18 or 18′ and 20 or 20′).

The primers 19 or 19′ and 21 or 21′ in the priming fluid may graft tothe second polymer layer 32′ that overlies the interstitial regions 30.Because the second polymer layer 32′ may not adhere to the polymer layer32, because amplification has been performed in the region 14, andbecause the functional groups of the underlying polymer layer 32 havebeen rendered non-functional, the primers 19 or 19′ and 21 or 21′ maynot graft to the exposed polymer layer 32.

As shown in FIG. 7E, the grafted primers 19 or 19′ and 21 or 21′ attachto the second polymer layer 32′ that overlies the interstitial regions30, which forms the region 16.

Additional amplification may then be performed. For example, bridgeamplification may be initiated from the cluster to the grafted primers19 or 19′ and 21 or 21′ in order to form a second cluster on at leastsome of the interstitial regions. During this round of amplification,the un-cleavable first template strands 40 loop over and hybridize toadjacent, complementary primers 21, 21′ while the cleavable secondtemplate strands 42 loop over and hybridize to adjacent, complementaryprimers 19, 19′. The respective strands 40, 42 are extended to form twonew double stranded loops. The process is repeated on each template copyby cycles of isothermal denaturation and amplification to create denseclonal clusters that grow out of the depressions 28 into theinterstitial regions 30. Each additional cluster of double strandedbridges is denatured, resulting in un-cleavable second template strands44 attached to the un-cleavable second primers 21, 21′ and cleavablefirst template strands 46 attached to the cleavable first primers 19,19′ as shown in FIG. 7F. Because the cleavable first template strands 46attached to the cleavable first primers 19, 19′ include the cleavagesite 22′ or 23, the cleavable first template strands 46 are cleavable.

It is to be understood that when the un-cleavable first template strands40 are forward strands, the un-cleavable second template strands 44 arereverse strands, and when the un-cleavable first template strands 40 arereverse strands, the un-cleavable second template strands 44 are forwardstrands. Similarly, when the cleavable first template strands 46 areforward strands, the cleavable second template strands 42 are reversestrands, and when the cleavable first template strands 46 are reversestrands, the cleavable second template strands 42 are forward strands.

The cleavable first and second template strands 46, 42 may then beremoved by introducing a chemical agent or an enzymatic cleaving agentdepending on the cleavage sites 22, 22′ or 22, 23. As shown in FIG. 7G,after cleavage, the un-cleavable first template strands 40 remain in theregion 14 in the depression 28, and the un-cleavable second templatestrands 44 remain in the region 16 on the interstitial regions 30. Inone example after cleavage is performed, the region 14 includes forwardstrands and the region 16 includes reverse strands.

Another example method for making the examples shown in FIGS. 5, 6A and6B is shown in FIGS. 8A through 8G. As shown in FIG. 8A, the methodutilizes the substrate 26 having a plurality of depressions 28 separatedby interstitial regions 30. In this example method, the polymer layer 32is deposited on the substrate 26 so that it is present in thedepressions 28 and on the interstitial regions 30. If it is desirablefor the regions 14 to be in the depressions 28, then the primers 18, 20or 18′, 20′ may be grafted using any of the examples disclosed herein.If it is desirable for the regions 16 to be in the depressions 28, thenthe primers 19, 21 or 19′, 21′ may be grafted using any of the examplesdisclosed herein. In the example shown in FIG. 8B, the primers 18, 20 or18′, 20′ are grafted to the polymer layer 32. It is to be understoodthat the primers 18, 20 or 18′, 20′ will graft to the polymer layer 32across the entire substrate 26, as shown in FIG. 8B.

In this example method, as shown at FIG. 8C, a protective coating 48 isthen deposited on the polymer layer 32 having the primer set 12A, 12B,12C, 12D grafted thereto. The protective coating 48 may be a lift-offresist. This type of protective coating 48 may be spun on, cured, andsubsequently removed at a desirable time in the process. The protectivecoating 48 may also be a water-soluble coating.

Etching may then be performed to expose those interstitial regions 30where it is desirable to generate the region 16 including the primer set12A′, 12B′, 12C′, 12D′. For example, an isolated section of thesubstrate surface S next to each depression 28 may be exposed viaetching to form the example shown in FIG. 5. For another example, asection of the substrate surface S that surrounds the entire depression28 may be exposed via etching to form the example shown in FIGS. 6A and6B. FIG. 8D illustrates one example of the substrate 26 after etching isperformed to expose the interstitial regions 30. In this example, plasmaetching may be performed with air or oxygen gas.

This example method may then involve silanizing the exposed portions ofthe substrate surface S. Silanization introduces an adhesion promotor tothe substrate surface S to help the second polymer layer 32′ adherethereto.

The second polymer layer 32′ is then deposited. As shown in FIG. 8E, thesecond polymer layer 32′ may be deposited on the exposed portions of thesubstrate surface S (e.g., at the interstitial regions 30) and on theprotective coating 48. In this example method, the polymer layer 32′ maybe the same polymer that is used in the polymer layer 32, or may be adifferent type of polymer than that used in the polymer layer 32.

As shown in FIG. 8F, the primers 19, 19′ and 21, 21′ of the secondprimer set 12A′, 12B′, 12C′, 12D′ may then be grafted to the polymerlayer 32′ to generate the regions 16. Grafting may be performed usingany of the methods described herein. In another example, the polymerlayer 32′ may be pre-grafted with the primers 19, 19′ and 21, 21′.

While not shown, it is to be understood that this example method maythen include removing the protective coating 48. As an example, alift-off method may be used to remove the protective coating 48 and anypolymer layer 32′ and primers 19, 19′ and 21, 21′ thereon. If theprotective coating 48 is water soluble, removal may involve dissolvingthe coating 48 in water, which will also remove any overlying polymerlayer 32′ and primers 19, 19′ and 21, 21′. Removal of the protectivecoating exposes all of the primer 18, 18′ and 20, 20′ at the regions 14and all of the primer 19, 19′ and 21, 21′ at the regions 16.

While not shown in FIG. 8A through FIG. 8F, it is to be understood thata plurality of library templates may then be introduced to the substrate26. Because the substrate 26 includes an array of regions 14 in thedepressions 28, multiple library templates are hybridized, for example,to one of two types of primers 18, 18′ or 20, 20′ immobilized therein.In this example, because the substrate 26 also includes an array ofregions 16 on at least some of the interstitial regions 30, multiplelibrary templates are hybridized, for example, to one of two types ofprimers 19, 19′ or 21, 21′ immobilized therein. Cluster generation maythen be performed as described herein. Cleavage of the cleavabletemplates may also be performed by cleaving the cleavable first primer19, 19′ and the cleavable second primer 20, 20′

FIG. 9 illustrates yet another example of the regions 14, 16 that arelocated in different portions of the depression 28. In this example, oneof the regions 14 or 16 is part of a bead 50 that is positioned in thedepression 28. The bead 50 includes a core structure 49 and the region16 at the surface of the core structure 49. In this example, the region16 may include functional group(s) inherently present at the surface ofthe core structure 49, or functional group(s) incorporated on thesurface of the core structure 49 through any suitable functionalizationtechnique (e.g., chemical reaction, coating the core structure 49 with areactive group-containing polymer, etc.).

While a single depression 28 is shown in FIG. 9, it is to be understoodthat in some examples, the substrate 26 includes a plurality ofdepressions 28 separated by interstitial regions 30; and each of thedepressions includes a first portion where the first region 14 islocated, and a second portion; and the flow cell further comprises abead 50 located in the second portion, wherein the second region 16 isat a surface of the bead. Several variations of this example are furtherdescribed herein in reference to FIGS. 11A through 20 in the section“Bead Based Flow Cell”.

FIG. 10 depicts still another example configuration for the regions 14,16. In this example, the regions 14, 16 are positioned on separatesubstrates 26, 26′. As such, this example of the flow cell includes afirst substrate 26, a first primer set 12A attached to the firstsubstrate 26, the first primer set including an un-cleavable firstprimer 18, 18′ and a cleavable second primer 20, 20′, a second substrate26′ opposed to the first substrate 26, and a second primer set 12A′attached to the second substrate 26′, the second primer set 12A′including a cleavable first primer 19, 19′ and an un-cleavable secondprimer 21, 21′. As one example of the method for making this flow cell,each of the substrates 26, 26′ may be coated with the polymer layer(e.g., 32, not shown in FIG. 10), the first primer set 12A (or 12B, 12C,or 12D) may be grafted to one of the substrates 26, and the secondprimer set 12A′ (or 12B′, 12C′, or 12D′) may be grafted to the other ofthe substrates 26′.

While shown on the respective substrate surfaces S, it is to beunderstood that the regions 14, 16 may alternatively be positioned indepressions 28 of the respective substrates 26, 26′. In this example,the polymer layer may be deposited in the depressions 28 and on theinterstitial regions 30, and then may be removed from the interstitialregions 30 via polishing. The first primer set 12A (or 12B, 12C, or 12D)may be grafted to the depressions 28 of one substrate 26, and the secondprimer set 12A′ (or 12B′, 12C′, or 12D′) may be grafted to thedepressions of the other substrate 26′.

In this example, it may be desirable for the substrates 26, 26′ to bewithin close proximity so that template seeding and clustering may beperformed successfully. In an example, “close proximity” means that thedistance between the two substrates 26, 26′ is about 100 μm or less. Inanother example, the distance between the two substrates 26, 26′ rangesfrom about 10 μm to about 90 μm.

Instead of being positioned on separate substrates 26, 26′, the firstand second regions 14, 16 and primer sets 12A, 12A′, or 12B, 12B′, or12C, 12C′, or 12D, 12D′ may be macro-separated on the substrate surfaceS or may be present in separate depressions 28 that are macro-separatedfrom each other. By macro-separated it is meant that the regions 14, 16are separated from each other by at least 5 μm. In one example, theregions 14, 16 are separated from each other by a distance ranging fromabout 5 μm to about 100 μm.

FIG. 33A depicts still another example configuration for the regions 14,16. In this example, the first and second functionalized layers 60, 64are integrated into the resin portion of the multi-layered substrate. Asshown in FIG. 33A, this example of the multi-layer substrate includes asupport 52; the first functionalized layer 60 on the support 52; thesecond functionalized layer 64 on the first functionalized layer 60; anda passivation layer 86 on the second functionalized layer 64.

In this example, the first and second functionalized layers 60, 64 maybe any nanoimprint lithography resin having or capable of havingintroduced thereto surface functional groups that can attach to therespective primer sets 12A, 12A′, or 12B, 12B′, or 12C, 12C′, or 12D,12D′. In one example, layer 60 may be functionalized with epoxy groupsand layer 64 may be functionalized with amine groups.

The resins of the layers 60, 64 may be the same if the respective primersets 12A, 12A′, or 12B, 12B′, or 12C, 12C′, or 12D, 12D′ are pre-graftedinto the layers 60, 64 before the multi-layer substrate shown in FIG.33A is formed. In these examples of this method, the first primer set12A, 12B, 12C, 12D (e.g., an un-cleavable first primer 18, 18′ and acleavable second primer 20, 20′) may be pre-grafted to the firstfunctionalized layer 60 before the first functionalized layer 60 isincorporated into the multi-layer substrate; and the second primer set12A′, 12B′, 12C′, 12D′ (e.g., a cleavable first primer 19, 19′ and anun-cleavable second primer 21, 21′) may be pre-grafted to the secondfunctionalized layer 66 before the second functionalized layer 64 isincorporated into the multi-layer substrate.

The layers (whether pre-grafted or not) may be deposited on the support52 using any of the examples disclosed herein.

When the primers are grafted post-imprinting, it is to be understoodthat the layers 60, 64 at least have different surface functional groupsthat can attach to the respective primer sets 12A, 12A′, or 12B, 12B′,or 12C, 12C′, or 12D, 12D′. Any of the resins disclosed herein may beused.

The passivation layer 86 may be any hydrophobic layer that can beimprinted. As examples, the passivation layer 86 is selected from thegroup consisting of a fluorinated polymer, a perfluorinated polymer, asilicon polymer, and a mixture thereof. As examples, the passivationlayer 86 may include an amorphous fluoropolymer (commercially availableexamples of which include those in the CYTOP® series from AGC Chemicals,which have one of the following terminal functional groups: A type:—COOH, M type: —CONH—Si(OR)_(n) or S type: —CF₃), apolytetrafluoroethylene (a commercially available example of which isTEFLON® from Chemours), parylen, a fluorinated hydrocarbon, afluoroacrylic copolymer (a commercially available example of whichincludes as FLUOROPEL® from Cytonix). The passivation layer 86 may bedeposited on the support 52 using any of the examples disclosed herein.

As shown in FIG. 33A, this example of the method includes imprinting themulti-layer substrate, thereby forming features (e.g., depressions 28)separated by interstitial regions 30 of the passivation layer 86,wherein a region 14, 16, respectively, of each the first and secondfunctionalized layers is exposed at each feature/depression 28. Theexposed regions 14, 16 of a single feature/depression 28 and thesurrounding interstitial regions 30 of the passivation layer 86 areshown from a top view in FIG. 33B. For each region 14, 16, thefeature/depression 28 has a slanted bottom or a step region, or someother variation in geometry that exposes both a portion of the layer 60and a portion of the layer 64 from the top of the feature/depression 28.

To imprint the multi-layer substrate, a nanoimprint lithography mold orworking stamp 56 is pressed against the layer of resin 54 to create animprint of the features in the layers 60, 64, 68. In other words, theeach of the layers 60, 64, 68 is indented or perforated by theprotrusions of the working stamp 56. The layers 60, 64, 68 may be thenbe cured with the working stamp 56 in place. Curing may be accomplishedby exposure to actinic radiation, such as visible light radiation orultraviolet (UV) radiation, or to radiation of a wavelength ranging fromabout 240 nm and 380 nm when a photoresist is used; or by exposure toheat when a thermal-curable resist is used. Curing may promotepolymerization and/or cross-linking. As an example, curing may includemultiple stages, including a softbake (e.g., to drive off solvent(s))and a hardbake. The softbake may take place at a lower temperature,ranging from about 50° C. to about 150° C. The duration of the hardbakemay last from about 5 seconds to about 10 minutes at a temperatureranging from about 100° C. to about 300° C. Examples of devices that canbe used for softbaking and/or hardbaking include a hot plate, oven, etc.

After curing, the working stamp 56 is released. This creates topographicfeatures, i.e., the depressions 28, in the layers 60, 64, 68.

As mentioned, the primers 18, 20 or 18′, 20′ (not shown in FIG. 33A orFIG. 33B) may be pre-grafted to the first functionalized layer 60, andthus are attached to the first functionalized region 14; and the primers19, 21 or 19′, 21′ (not shown in FIG. 33A or FIG. 33B) may bepre-grafted to the second functionalized layer 64, and thus are attachedto the second functionalized region 16. In these examples, additionalprimer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ and/or the primers 19,21 or 19′, 21′ are not pre-grafted, respectively, to the firstfunctionalized layer 60 and the second functionalized layer 64. In theseexamples, the regions 14, 16 have different functional groups, and theprimers 18, 20 or 18′, 20′ and primers 19, 21 or 19′, 21′ may be graftedafter imprinting.

When grafting is performed after imprinting, grafting may beaccomplished using any grafting technique disclosed herein. With any ofthe grafting methods, the primers 18, 20 or 18′, 20′ react with reactivegroups of the region 14 or the primers 19, 21 or 19′, 21′ react withreactive groups of the region 16, and have no affinity for thepassivation layer 86.

As mentioned, some examples of the depression 28 may be in the form oftrenches. FIG. 34S depicts (from a top view) still another exampleconfiguration for the regions 14, 16, where they are formed in trenches28″.

As shown at FIG. 34A, the multi-layered substrate includes the(patterned) resin 54′ on the support 52. The trenches 28″ defined in thepatterned resin 54′ (e.g., an NIL resin, SiO₂, etc.) are adjacent tointerstitial regions 30, which separate adjacent trenches 28″ from oneanother (see, e.g., FIG. 34H, which illustrates a top view of multipletrenches 28″).

As shown in FIG. 34B, a sacrificial layer 84 is applied on the resin 54′(e.g., interstitial regions 30 and in the trench 28″). Any material maybe used as the sacrificial layer 84 that has an etch differentialrelative to the resin 54′ and relative to a second sacrificial layer 88that is used. In an example, the sacrificial layer 84 is silicon,aluminum, or chromium.

As shown in FIG. 34C, the sacrificial layer 84 is removed from theportions of the resin 54′. In this example, the sacrificial layer 84 maybe etched such that a region of the sacrificial material 84″ remainsdirectly adjacent to each sidewall 90 of each of the trenches 28″. Afteretching, interstitial regions 30 and a bottom portion of each trench 28″is exposed.

As shown in FIG. 34D, a second sacrificial layer 88 is applied on theregions of the sacrificial material 84″ and on any exposed areas of theresin 54′ (e.g., interstitial regions 30 and in the bottom portion oftrench 28″). Any material may be used as the second sacrificial layer 88that has an etch differential relative to the resin 54′ and relative toa second sacrificial layer 84 that is used. For example, if layer 84 issilicon, then layer 88 may be aluminum or chromium.

The sacrificial layers 84, 88 may be applied using any of the depositiontechniques described herein.

As shown in FIG. 34E, the second sacrificial layer 88 is removed fromthe regions of the sacrificial material 84″ and from portions of theresin 54′. In this example, the second sacrificial layer 88 may beetched such that a region of the second sacrificial material 88″ remainsdirectly adjacent to each of the sacrificial material regions 84″. Inthis example, after etching, interstitial regions 30 and another(smaller) bottom portion of each trench 28″ is exposed.

As shown in FIG. 34F (depicting a top view), a material 92 is depositedto fill any spaces between the second sacrificial material regions 84″.In other words, the additional material 92 covers the bottom portion ofeach trench 28″, and completely separates the regions of the secondsacrificial material 88′. The additional material 92 may be the samematerial as the resin 54′. For example, if silicon dioxide is used asthe resin 54′, then the additional material 92 may be silicon dioxide.The additional material 92 helps to define new tranches 28′″ (in whichthe regions 14,1 6 will be defined), which are smaller than the trenches28″.

In this example of the method, as shown in FIG. 34G, polishing may beperformed to remove the additional material 92 from the interstitialregions 30′.

FIG. 34H depicts a top view of the trenches 28′″ filled with the twosacrificial materials/layers 84″, 88′. As shown each of the sacrificialmaterials/layers 84″, 88′ extends the length of each trench 28′″.

FIG. 34I depicts a cross-sectional view of one of the trenches 28′″filled with the two sacrificial materials/layers 84″, 88′.

As shown in FIG. 34J, the sacrificial material region 84″ is removedfrom a portion of each of the trenches 28′″. This exposes anarea/portion 76 where the first functionalized region 14 is to beformed. The sacrificial material region 84″ may be etched, and thisetching process does not affect the resin 54′ or the second sacrificialmaterial region 88′ due to the different etch rates.

In FIG. 34K, a first functionalized layer 60 is applied on theinterstitial regions 30, the area 76, and the second sacrificialmaterial region 88′. The first functionalized layer 60 may be any of theexamples disclosed herein and may be deposited using any of thetechniques described herein.

In FIG. 34KL and FIG. 34M, the first functionalized layer 60 is thenpatterned to form a first functionalized region (region 14) covered by aphotoresist 62 in the area/portion 76. The photoresist 62 and theunderlying first functionalized layer 60 may be polished or etched away,using the interstitial regions 30 and the second sacrificial materialregion 88′ as an etch stop. This forms the first functionalized region14 covered by the photoresist 62 in the area/portion 76 of the trench28″′.

As shown in FIG. 34N, the second sacrificial material region 88′ isremoved from a portion of each of the trenches 28″′. This exposes anarea/portion 78 where the second functionalized region 16 is to beformed. The second sacrificial material region 88′ may be etched, andthis etching process does not affect the resin 54′ or the photoresist 62due to the different etch rates. The exposed portion of the firstfunctionalized layer 60 may be removed during this etching process.

In FIG. 34O, a second functionalized layer 64 is applied on theinterstitial regions 30, the area 78, the photoresist 62, and anyexposed portion of the region 14. The second functionalized layer 64 maybe any of the examples disclosed herein and may be deposited using anyof the techniques described herein.

In FIG. 34P, the photoresist 62 may then be lifted off, which removesany of the second functionalized layer 64 thereon. In any of theexamples disclosed herein, sonication may be performed to improve theefficiency of the photoresist stripping or lift off process.

In FIG. 34Q, the interstitial regions 30 may be polished to remove anyof the second functionalized layer 64 thereon. As shown in FIG. 34Q, theregions 14, 16 are formed in respective regions of the trench 28′″. Itis to be understood that the regions 14, 16 extend the length of thetrenches 28′″ (as shown in FIG. 34R). This configuration of the regions14, 16 may be used for simultaneous paired end read sequencing using theprimer sets 12A, 12A′, or 12B, 12B′, or 12C, 12C′, or 12D, 12D′disclosed herein.

In FIG. 34R, a second photoresist 62′ is applied (and exposed anddeveloped) to form a pattern of spatially separated stripes that are atleast substantially perpendicular to the trenches 28′″. This patternleaves portions of the first functionalized regions 14 and the secondfunctionalized regions 14 that are exposed between the spatiallyseparated stripes, as shown in FIG. 34R. The portions of the firstfunctionalized regions 14 and the second functionalized regions 16 thatare exposed between the spatially separated stripes are removed (e.g.,via etching), and then the spatially separated stripes (secondphotoresist 62′) are removed (e.g., via etching). The regions 14, 16underlying the spatially separated stripes (second photoresist 62′)remain intact after removal of the spatially separated stripes (secondphotoresist 62′), as shown in FIG. 34S. In this example, the respectiveregions 14, 16 are isolated pairs along the trenches 28′″.

In some examples, the primers 18, 20 or 18′, 20′ (not shown in FIG. 34Athrough FIG. 34S) may be pre-grafted to the first functionalized layer60, and thus are attached to the first functionalized regions 14.Similarly, the primers 19, 21 or 19′, 21′ (not shown in FIG. 34A throughFIG. 34S) may be pre-grafted to the second functionalized layer 64, andthus are attached to the second functionalized regions 16. In theseexamples, additional primer grafting is not performed.

In other examples, the primers 18, 20 or 18′, 20′ are not pre-grafted tothe first functionalized layer 60. In these examples, the primers 18, 20or 18′, 20′ may be grafted after the first functionalized layer 60 isapplied (e.g., at FIG. 34K). If the regions 14, 16 have differentfunctional groups, the primers 18, 20 or 18′, 20′ may be grafted at theend of the method (e.g., at FIG. 34S), because they will not graft tothe surface functional groups of the regions 16.

Similarly, the primers 19, 21 or 19′, 21′ may not be pre-grafted to thesecond functionalized layer 64. In these examples, the primers 19, 21 or19′, 21′ may be grafted after the second functionalized layer 64 isapplied (e.g., at FIG. 34O). If the regions 14, 16 have differentfunctional groups, the primers 19, 21 or 19′, 21′ may be grafted at theend of the method (e.g., at FIG. 34S), because they will not graft tothe surface functional groups of the regions 14.

When grafting is performed during the method, grafting may be performedusing any example disclosed herein. With any of the grafting methods,the primers 18, 20 or 18′, 20′ react with reactive groups of the region14 or the primers 19, 21 or 19′, 21′ react with reactive groups of theregion 16, and have no affinity for the resin 54′.

In still another example of the flow cell, the regions 14, 16 may beformed on respective functionalized support structures 93, 94, as shownin FIG. 35. The functionalized support structures 93, 94 have surfacefunctional groups that they can attach the respective primer sets 12A,12A′, or 12B, 12B′, or 12C, 12C′, or 12D, 12D′ disclosed herein. Anyexample of the core structure 49 disclosed herein may be used for thefunctionalized support structures 93, 94. Any of the surface functionalgroups disclosed herein that are capable of attaching the differentprimer sets 12A, 12A′, or 12B, 12B′, or 12C, 12C′, or 12D, 12D′ may alsobe used. The functionalized support structure 93, 94 may be formed, forexample, using the method(s) disclosed herein for forming thefunctionalized bead 50 (see the section “Bead Based Flow Cell”).

In some examples, the functionalized support structures 93, 94 may alsohave different shapes. In some examples, the respective shapescorrespond with, respectively, the shapes of the capture sites 95, 96(shown in FIG. 36A, FIG. 36B, FIG. 37A, and FIG. 37B) on or defined in asurface S of the substrate (which may be a single layer or multi-layersubstrate as described herein). For example, the functionalized supportstructures 93, 94 may respectively have the same shape as well capturesites 95, 96 (described below). The different shapes will aid in thefunctionalized support structure 93 becoming physically entrapped in thecomplementary shaped well capture site 95 and in the functionalizedsupport structure 94 becoming physically entrapped in the complementaryshaped well capture site 96.

In some examples, the functionalized support structures 93, 94 may alsohave different capture chemistry. The capture chemistry is the chemistrythat enables the structures 93, 94 to attach to a desirable location onthe substrate surface S. In some examples, the capture chemistry of thefunctionalized support structures 93, 94 respectively corresponds withthe capture chemistry of the capture sites 95, 96 that is to receive therespective functionalized support structure 93, 94. For example, thefunctionalized support structure 93 and the capture site 95 may eachinclude a member of one type of receptor-ligand binding pair, while thefunctionalized support structure 94 and the capture site 96 may eachinclude a member of a different type of receptor-ligand binding pair.The different chemistries can help to endure that the functionalizedsupport structures 93, 94 (and the respective regions 14, 16) arecaptures at desirable regions on the substrate surface S.

The capture sites 95, 96 are physically and/or chemically capable ofimmobilizing the respective functionalized support structure 93, 94 onthe substrate 26 (or resin 54′ of a multi-layer substrate). The capturesites 95, 96 may be positioned at any suitable location where it isdesirable to have adjacent regions 14, 16. The position of the capturesites 95, 96 across the substrate 26 may be uniform (see FIGS. 36A and36B) or may be non-uniform. The capture sites 95, 96 may have anysuitable shape, geometry and dimensions, which may depend, at least inpart, on the configuration of the capture sites 95, 96 (e.g., a patch, awell, a protrusion, etc.), and the type of functionalized supportstructure 93, 94 that is to be captured by the capture sites 95, 96.

In some examples, the capture sites 95, 96 are different chemicalcapture agents that are applied on a portion of the substrate surface S.Any examples of the chemical capture agent disclosed herein may be used.In one example, the respective chemical capture agents may be depositedin the desirable locations using microcontact printing, or anothersuitable technique.

In other examples, the capture sites 95, 96 include respective wellsthat are defined in the surface S of the substrate 26 (or resin 54′).The wells may be formed using etching or imprinting depending upon thesubstrate (e.g., single or multi-layer) that is used. The wells may haveany suitable shape and geometry, such as those set forth herein for thedepressions 28.

In some examples, the wells do not have an additional chemical captureagent added thereto. In these examples, the opening dimensions enablethe respective functionalized support structure 93, 94 to self-assembleinto the corresponding wells (e.g., based on shape). In other examples,the wells do have respective chemical capture agents added thereto.

Other examples of the capture sites 95, 96 include the well and acapture bead having a chemical capture agent on a surface thereof. Thecapture bead may be sized to fit into the wells. In some examples, thecapture beads may be co-planar with or extend slightly above theadjacent interstitial regions 30, 30′ so that the functionalized supportstructure 93, 94 that ultimately attaches thereto is not confined withinthe well. In an example, the capture bead is selected from the groupconsisting of silicon dioxide, a superparamagnetic material,polystyrene, and an acrylate. Any examples of the chemical capture agentdisclosed herein may be used on the surface of the capture bead, and maybe coated on the capture bead before it is introduced into the well. Theconfiguration of the wells and beads of these capture sites 95, 96 maybe such that the functionalized support structure 93, 94 (when attached)form regions 14, 16 that are adjacent to one another.

The depth of the capture sites 95, 96 well may vary depending uponwhether the chemical capture agent is to be introduced thereto andwhether the capture bead is to be introduced thereto. The depth may beselected at least to accommodate these materials (i.e., the material iscontained within the wells). In an example, the depth of the well rangesfrom about 1 nm to about 5 microns.

As another example, the capture sites 95, 96 include protrusions thatare defined in the substrate 26 (or in the resin 54′). The protrusionsare three-dimensional structures that extend outward (upward) from anadjacent surface. The protrusions may be generated via etching,photolithography, imprinting, etc.

While any suitable three-dimensional geometry may be used for theprotrusion capture sites 95, 96, a geometry with an at leastsubstantially flat top surface may be desirable. Example protrusiongeometries include a sphere, a cylinder, a cube, polygonal prisms (e.g.,rectangular prisms, hexagonal prisms, etc.), or the like.

Different chemical capture agents may be applied on the top surface ofthe respective protrusion capture sites 95, 96. Any examples of thechemical capture agent disclosed herein may be used, and any depositiontechnique may be used to apply the chemical capture agent to the topsurface of the protrusions.

It is to be understood that while the capture sites 95, 96 have beendescribed as both being capture agents, wells, etc., any combination ofthe types of capture sites 95, 96 may be used together (e.g., captureagent and well) on the flow cell.

In FIG. 36A, the capture sites 95, 96, on the surface S have differentshapes. When the functionalized support structure 93, 94 havingrespectively corresponding shapes are loaded into the flow cell (FIG.36B), the functionalized support structure 93, 94 self-assemble, eitherby physical exclusion and/or by the capture chemistry, so that thefunctionalized support structures 93 attach to the capture sites 95 andthe functionalized support structure 94 attach to the capture sites 96.

In FIG. 37A, the capture sites 95, 96, on the surface S also havedifferent shapes, but are arranged differently than the example shown inFIG. 36A. When the functionalized support structure 93, 94 havingrespectively corresponding shapes are loaded into the flow cell (FIG.37B), the functionalized support structure 93, 94 self-assemble, eitherby physical exclusion and/or by the capture chemistry, so that thefunctionalized support structures 93 attach to the capture sites 95 andthe functionalized support structure 94 attach to the capture sites 96.

Both configurations shown in FIG. 36B and FIG. 37B result in an array ofregions 14, 16 in isolated positions across the substrate surface S.

FIG. 2 through FIG. 10, FIG. 33A, FIG. 34S, FIG. 36B, and FIG. 37Billustrate different configurations for the regions 14, 16 of the flowcell without a lid bonded to the substrate 26 (or the resin 54′). Whilenot shown, it is to be understood that the flow cells may have the lidbonded to at least a portion of the interstitial region 30, 30′. In someexamples, the lid may be bonded before or after grafting of the primersets 12A, 12A′, or 12B, 12B′, or 12C, 12C′, or 12D, 12D′. When the lidis bonded prior to primer grafting, it is to be understood that a flowthrough process may be used for grafting. In the flow through process,the primer solution or mixture may be introduced into a flow channel(s)(defined between the lid and the interstitial region 30, 30′) throughrespective input port(s) (not shown), may be maintained in the flowchannel(s) for a time sufficient (i.e., an incubation period) for theprimer(s) 18, 18′, 20, 20′, 19, 19′, 21, 21′ to attach to the respectiveregions 14, 16, and then may be removed from respective output port(s)(not shown). After primer 18, 18′, 20, 20′, 19, 19′, 21, 21′ attachment,the additional fluid(s) may be directed through the flow channel(s) towash the now functionalized depressions and the flow channel(s).

The lid may be positioned on the interstitial region 30 so that itdefines a single flow channel or multiple, fluidically separated flowchannels.

The lid may be any material that is transparent to an excitation lightthat is directed toward the substrate 26. As examples, the lid may beglass (e.g., borosilicate, fused silica, etc.), plastic, or the like. Acommercially available example of a suitable borosilicate glass is D263®, available from Schott North America, Inc. Commercially availableexamples of suitable plastic materials, namely cyclo olefin polymers,are the ZEONOR® products available from Zeon Chemicals L.P.

In some examples, the lid may be integrally formed with sidewall(s) thatcorrespond with the shape of the portion of the interstitial region 30to which it will be bonded. For example, a recess may be etched into atransparent block to form a substantially planar (e.g., top) portion andsidewall(s) extending from the substantially planar portion. When theetched block is mounted to the interstitial region 30, the recess maybecome the flow channel. In other examples, the sidewall(s) and the lidmay be separate components that are coupled to each other. For example,the lid may be a substantially rectangular block having an at leastsubstantially planar exterior surface and an at least substantiallyplanar interior surface that defines a portion (e.g., a top portion) ofthe flow channel (once bonded to the portion of the interstitial region30). The block may be mounted onto (e.g., bonded to) the sidewall(s),which are bonded to the portion of the interstitial region 30 and formsidewall(s) of the flow channel. In this example, the sidewall(s) mayinclude any of the materials set forth herein for the spacer layer(described below).

The lid may be bonded using any suitable technique, such as laserbonding, diffusion bonding, anodic bonding, eutectic bonding, plasmaactivation bonding, glass frit bonding, or others methods known in theart. In an example, a spacer layer may be used to bond the lid to theportion of the interstitial region 30. The spacer layer may be anymaterial that will seal at least some of the interstitial regions 30 andthe lid together.

In one example, the spacer layer may be a radiation-absorbing materialthat absorbs radiation at a wavelength that is transmitted by the lidand/or the substrate 26 (or, for example, a patterned resin of thesubstrate 26). The absorbed energy, in turn, forms the bond between thespacer layer and the lid and between the spacer layer and the substrate26. An example of this radiation-absorbing material is black KAPTON®(polyimide containing carbon black) from DuPont (USA), which absorbs atabout 1064 nm. It is to be understood that polyimide could be usedwithout the addition of carbon black, except that the wavelength wouldhave to be altered to one that is significantly absorbed by the naturalpolyimide material (e.g., 480 nm). As another example, polyimide CEN JPcan be bonded when irradiated with light at 532 nm. When the spacerlayer is the radiation-absorbing material, the spacer layer may bepositioned at an interface between the lid and the portion of theinterstitial region 30 so that the spacer layer contacts the desiredbonding region. Compression may be applied (e.g., approximately 100 PSIof pressure) while laser energy at a suitable wavelength is applied tothe interface (i.e., the radiation-absorbing material is irradiated).The laser energy may be applied to the interface both from the top andfrom the bottom in order to achieve suitable bonding.

In another example, the spacer layer may include a radiation-absorbingmaterial in contact therewith. The radiation-absorbing material may beapplied at the interface between the spacer layer and the lid as well asat the interface between the spacer layer and the portion of theinterstitial region 30. As an example, the spacer layer may be polyimideand the separate radiation-absorbing material may be carbon black. Inthis example, the separate radiation-absorbing material absorbs thelaser energy that forms the bonds between the spacer layer and the lidand between the spacer layer and the portion of the interstitial region30. In this example, compression may be applied at the respectiveinterfaces while laser energy at a suitable wavelength is applied to theinterfaces (i.e., the radiation-absorbing material is irradiated).

Simultaneous Paired-End Sequencing Method

Any examples of the flow cell including the primer sets 12A, 12A′, or12B, 12B′, or 12C, 12C′, or 12D, 12D′ described herein may be used insimultaneous paired-end sequencing, where the forward and reversestrands (e.g., strands 40 and 44) are read simultaneously.

Once the clusters of un-cleavable first template strands 40 aregenerated in the regions 14 and the clusters of un-cleavable secondtemplate strands 44 are generated in the region 16 (described inreference to FIG. 7C through FIG. 7G), the free ends may be blocked toprevent undesirable priming. It is to be understood that the free endsof any primers that remain may also be blocked.

In these methods, an incorporation mix may be added, which includessequencing primers that are capable of respectively hybridizing to theun-cleavable first template strands 40 and the un-cleavable secondtemplate strands 44. The extension of the sequencing primers along therespective template strands 40, 44 is monitored to determine thesequence of nucleotides in the template strands 40, 44. The underlyingchemical process can be polymerization (e.g., catalyzed by a polymeraseenzyme) or ligation (e.g., catalyzed by a ligase enzyme). In aparticular polymerase-based process, fluorescently labeled nucleotidesare added to the respective sequencing primers in a template dependentfashion such that detection of the order and type of nucleotides addedto the respective sequencing primers can be used to determine thesequence of the template. For example, to initiate a first sequencing bysynthesis cycle, one or more labeled nucleotides, DNA polymerase, etc.,may be delivered into/through the flow cell, where sequencing primerextension causes a labeled nucleotide to be incorporated into a nascentstrand that is complementary to the respective template 40, 44. Theincorporation events can be detected in tandem through an imaging eventwithout substantial physical overlap of the fluorescent signalsgenerated at the respective templates 40, 44. This allows forsimultaneous base calling at the respective templates 40, 44. During animaging event, an illumination system (not shown) may provide anexcitation light to the flow cell, and images may be captured andanalyzed. As examples, illumination may be accomplished with a laser,light emitting diode, planar waveguide, or the like.

In some examples, the fluorescently labeled nucleotides can furtherinclude a reversible termination property that terminates further primerextension once a nucleotide has been added to the respective templates40, 44. For example, a nucleotide analog having a reversible terminatormoiety can be added to the templates 40, 44 such that subsequentextension cannot occur until a deblocking agent is delivered to removethe moiety. Thus, for examples that use reversible termination, adeblocking reagent can be delivered to the flow cell, etc. (afterdetection occurs).

Wash(es) may take place between the various fluid delivery steps. Thesequencing cycle can then be repeated n times to extend the template byn nucleotides, thereby detecting a sequence of length n.

Paired-end sequencing allows users to sequence both ends of a fragmentand generate high-quality, alignable sequence data. Paired-endsequencing facilitates detection of genomic rearrangements andrepetitive sequence elements, as well as gene fusions and noveltranscripts. While one example paired-end sequencing method has beendescribed in detail, it is to be understood that the flow cellsdescribed herein may be utilized with other sequencing protocol, forgenotyping, or in other chemical and/or biological applications. In yetanother example, the flow cells disclosed herein may be used for on-celllibrary generation.

Kits

Any example of the flow cells described herein may be part of a kit.

Some examples of the kit include the flow cell, a template mix/fluid,and an incorporation mix. These examples of the flow cell have bothprimer sets 12A, 12A′, or 12B, 12B′, or 12C, 12C′, or 12D, 12D′ attachedthereto. The template mix/fluid includes the template to be sequenced,and the incorporation mix includes sequencing primers that are capableof respectively hybridizing to the un-cleavable first template strands40 and the un-cleavable second template strands 44 (formed using thetemplate).

Other examples of the kit include the flow cell and the priming fluiddescribed herein. These examples of the flow cell have one primer set12A, 12B, 12C, or 12D attached thereto, and the priming fluid may beused to introduce the other primer set 12A′, 12B′, 12C′, or 12D′. In oneexample of the kit, the flow cell includes a substrate 26 includingdepressions 28 separated by interstitial regions 30, a first polymerlayer 32 in each of the depressions, wherein some functional groups ofthe first polymer layer are capped, a first primer set 12A, 12B, 12C, or12D attached to other functional groups of first polymer layer 32 ineach of the depressions 28, and a second polymer layer 32′ on theinterstitial regions 30; and the priming fluid includes a fluid carrier,and a second primer set 12A′, 12B′, 12C′, or 12D′ that is different fromthe first primer set 12A, 12B, 12C, or 12D. This example kit may alsoinclude the template mix and the incorporation mix.

Bead Based Flow Cell

FIG. 9 depicts one configuration for the flow cell, where one of theregions 14 or 16 is part of a bead 50. This section describes variousexamples and methods for this flow cell configuration.

Examples of this flow cell 10A (FIGS. 13D and 14), 10B (FIGS. 15D and16), 10C (FIGS. 17D and 18), 10D (FIGS. 19D and 20) disclosed hereininclude a support 52 and a patterned resin 54′, 54″ on the support 52,the patterned resin including depressions 28A, 28B or 28C separated byinterstitial regions 30. FIGS. 11A through 11D together depict anexample of a method for patterning a resin 54 to form the depressions28A, 28B or 28C. More specifically, FIGS. 11A through 11C depict theformation of the depressions 28A, 28B, and FIGS. 11A, 11B, and 11Ddepict the formation of the depressions 28C.

FIG. 11A depicts a support 52, and FIG. 11B depicts a resin 54 depositedon the support 52. Any example of the substrate 12 described herein maybe used for the support 52.

Some examples of suitable resins 54 are selected from the groupconsisting of a polyhedral oligomeric silsesquioxane resin (POSS)-basedresin, an epoxy resin, a poly(ethylene glycol) resin, a polyether resin,an acrylic resin, an acrylate resin, a methacrylate resin, andcombinations thereof. While several examples have been provided, it isbelieved that any resin that can be cured may be used.

As used herein, the terms “polyhedral oligomeric silsesquioxane” (POSS)and “POSS-based resin” refers to a chemical composition that is a hybridintermediate (RSiO_(1.5)) between that of silica (SiO₂) and silicone(R₂SiO). An example of POSS can be that described in Kehagias et al.,Microelectronic Engineering 86 (2009), pp. 776-778, which isincorporated by reference in its entirety. The composition is anorganosilicon compound with the chemical formula [RSiO_(3/2)]_(n), wherethe R groups can be the same or different. The composition may compriseone or more different cage or core structures as monomeric units. Insome instances, the structure includes the following polyoctahedral cageor core structure. In some instances, the polyhedral structure may be aT₈ structure, such as:

and represented by:

This monomeric unit typically has eight arms of functional groups R₁through R₈.

The monomeric unit may have a cage structure with 10 silicon atoms and10 R groups, referred to as T₁₀, such as:

or may have a cage structure with 12 silicon atoms and 12 R groups,referred to as T₁₂, such as:

The POSS-based material may include T₆, T₁₄, or T₁₆ cage structures. Theaverage cage content can be adjusted during the synthesis, and/orcontrolled by purification methods, and a distribution of cage sizes ofthe monomeric unit(s) may be used in the examples disclosed herein. Asexamples, any of the cage structures may be present in an amount rangingfrom about 30% to about 100% of the total POSS monomeric units used. ThePOSS-based material may be a mixture of cage structures along with openand partially open cage structures. Thus, a POSS-based resin precursoror resin may include epoxy POSS materials, which may be a mixture ofsilsesquioxane configurations. For example, any POSS material describedherein may be a mixture of discrete POSS cages and non-discretesilsesquioxane structures and/or incompletely condensed, discretestructures, such as polymers, ladders, and the like. The partiallycondensed materials would therefore include epoxy R groups as describedherein at some silicon vertices, but some silicon atoms would not besubstituted with the R groups and could be substituted instead with OHgroups. In some examples, the POSS materials comprise a mixture ofvarious forms, such as:

Condensed Cages

Incompletely Condensed Cages

Non-Cage Content Large & Ill-Defined Structure

and/or

In some of the examples disclosed herein, at least one of R₁ through R₈or R₁₀ or R₁₂ comprises an epoxy, and thus the POSS is referred to as anepoxy POSS. In some examples, a majority of the arms, such as the eight,ten, or twelve arms, or R groups, comprise epoxy groups. In otherexamples, R₁ through R₈ or R₁₀ or R₁₂ are the same, and thus each of R₁through R₈ or R₁₀ or R₁₂ comprises an epoxy group. In still otherexamples, R₁ through R₈ or R₁₀ or R₁₂ are not the same, and thus atleast one of R₁ through R₈ or R₁₀ or R₁₂ comprises epoxy and at leastone other of R₁ through R₈ or R₁₀ or R₁₂ is a non-epoxy functionalgroup. The non-epoxy functional group may be (a) a reactive group thatis orthogonally reactive to an epoxy group (i.e., reacts under differentconditions than an epoxy group), that serves as a handle for couplingthe resin to an amplification primer, a polymer, or a polymerizationagent; or (b) a group that adjusts the mechanical or functionalproperties of the resin, e.g., surface energy adjustments. In someexamples, the non-epoxy functional group is selected from the groupconsisting of an azide/azido, a thiol, a poly(ethylene glycol), anorbornene, a tetrazine, an amino, a hydroxyl, an alkynyl, a ketone, analdehyde, an ester group, an alkyl, an aryl, an alkoxy, and a haloalkyl.In some aspects, the non-epoxy functional group is selected to increasethe surface energy of the resin. In these other examples, the ratio ofepoxy groups to non-epoxy groups ranges from 7:1 to 1:7, or 9:1 to 1:9,or 11:1 to 1:11. In any of the examples, disubstituted ormonosubstituted (terminal) epoxy group(s) allow the monomeric unit topolymerize into a cross-linked matrix upon initiation using ultraviolet(UV) light and an acid. In some aspects, the epoxy POSS comprisesterminal epoxy groups. An example of this type of POSS is glycidyl POSShaving the structure:

An epoxy POSS may also be a modified epoxy POSS, that includes acontrolled radical polymerization (CRP) agent and/or another functionalgroup of interest incorporated into the resin or core or cage structureas one or more of the functional group R₁ through R₈ or R₁₀ or R₁₂.

In other of the examples of the POSS-based resin disclosed herein, eachof R₁ through R₈ or R₁₀ or R₁₂ comprises any non-epoxy group, such as anacrylate, methacrylate, ethylene glycol, or a short polyethylene glycolchain (up to 50 repeat units). Any non-epoxy POSS monomer that can bepolymerized radically may be used. An example is a methacryl POSS cagemixture having the following structure:

Another example is a PEG-POSS cage mixture having the followingstructure:

In the PEG-POSS example, the end methyl group (CH₃) of the R group maybe replaced with X, where X is an acryl group, a methacryl group, oranother suitable end group.

Other resins 54 may also be used. Examples include (non-POSS) epoxyresins, poly(ethylene glycol) resins, polyether resins (which may bering opened epoxies), acrylic resins, acrylate resins, methacrylateresins, and combinations thereof. As examples, a resin including epoxyand acrylate monomers may be used, or a resin including epoxy andethylene monomers may be used.

Another example of a suitable resin 54 is an amorphous fluoropolymer. Anexample of a commercially available amorphous (non-crystalline)fluoropolymer is CYTOP® from Bellex).

As shown in FIG. 11B, the resin 54 is deposited on the support 52. In anexample, deposition of the resin 54 involves chemical vapor deposition,dip coating, dunk coating, spin coating, spray coating, puddledispensing, ultrasonic spray coating, doctor blade coating, aerosolprinting, screen printing, microcontact printing, or inkjet printing.

The deposited resin 54 is then patterned, using any of the patterningtechniques mentioned herein. In the example shown in FIG. 11B,nanoimprint lithography is used to pattern the resin 54. After the resin54 is deposited, it may be soft baked to remove excess solvent. Ananoimprint lithography mold or working stamp 56 is pressed against thelayer of resin 54 to create an imprint on the resin 54. In other words,the resin 54 is indented or perforated by the protrusions of the workingstamp 56. The resin 54 may be then be cured with the working stamp 56 inplace. Curing may be accomplished by exposure to actinic radiation, suchas visible light radiation or ultraviolet (UV) radiation, or toradiation of a wavelength ranging from about 240 nm and 380 nm when aphotoresist is used; or by exposure to heat when a thermal-curableresist is used. Curing may promote polymerization and/or cross-linking.As an example, curing may include multiple stages, including a softbake(e.g., to drive off solvent(s)) and a hardbake. The softbake may takeplace at a lower temperature, ranging from about 50° C. to about 150° C.The duration of the hardbake may last from about 5 seconds to about 10minutes at a temperature ranging from about 100° C. to about 300° C.Examples of devices that can be used for softbaking and/or hardbakinginclude a hot plate, oven, etc.

After curing, the working stamp 56 is released. This creates topographicfeatures, i.e., the depressions 28A and 28B or 28C, in the resin 54. Asshown in FIG. 11C, the resin 54 having the depressions 28A and 28Bdefined therein is referred to as the patterned resin 54′. As shown inFIG. 11D, the resin 54 having the depressions 28C defined therein isreferred to as the patterned resin 54″. The patterned resin 54′, 54″ maybe subject to further hard baking to complete the cure and to lock inthe imprinted topography. In some examples, the hard baking may beperformed at a temperature ranging from about 60° C. to about 300° C.

The depressions 28A and 28B shown in FIG. 11C have different sizes. Inthis example, “different sizes” means that some of the depressions 28Bhave smaller opening dimensions than some other of the depressions 28A.In this example, the “opening dimension” refers to the area occupied byeach depression opening on the patterned resin 54′ and/or the diameterof each depression opening on the patterned resin 54′. In the exampleshown in FIG. 11C, the area and diameter of the opening of each of thedepressions 28B is smaller than the area and diameter of the opening ofeach of the depressions 28A. The area and diameter of the opening of thelarger depressions 28A depend upon the particle size (e.g., diameter) ofthe beads 50 to be introduced thereto. The area and diameter of theopening of the smaller depressions 28B are smaller than the particlesize of the beads 50. These opening dimensions enable the beads 50 toself-assemble into the depressions 28A and not the depressions 28B bysize exclusion.

The depressions 28C shown in FIG. 11D include two portions 34, 34′ thatare interconnected, but which have different sizes. In this example,“different sizes” means that the portion 34′ of each of the depressions28C has smaller opening dimensions than the portion 34. Also in thisexample, the “opening dimension” refers to the area occupied by eachportion opening on the patterned resin 54″ and/or the diameter of eachportion opening on the patterned resin 54″. In the example shown in FIG.11D, the area and diameter of the opening of each of the portions 34′ issmaller than the area and diameter of the opening of each of theportions 34. The area and diameter of the opening of the larger portions34 depend upon the particle size (e.g., diameter) of the beads 50 to beintroduced thereto. The area and diameter of the opening of the smallerportions 34′ are smaller than the particle size of the beads 50. Theseopening dimensions enable the beads 50 to self-assemble into theportions 34 and not the portions 34′ by size exclusion.

Examples of the area for each depression opening or portion opening on asurface can be at least about 1×10⁻³ μm², at least about 1×10⁻² μm², atleast about 0.1 μm², at least about 1 μm², at least about 10 μm², atleast about 100 μm², or more. Alternatively or additionally, the areacan be at most about 1×10³ μm², at most about 100 μm², at most about 10μm², at most about 1 μm², at most about 0.1 μm², at most about 1×10⁻²μm², or less. In some instances, the diameter of each depression 28A,28B or portion 34, 34′ can be at least about 50 nm, at least about 0.1μm, at least about 0.5 μm, at least about 1 μm, at least about 10 μm, atleast about 100 μm, or more. Alternatively or additionally, the diametercan be at most about 1×10³ μm, at most about 100 μm, at most about 10μm, at most about 1 μm, at most about 0.5 μm, at most about 0.1 μm, orless (e.g., about 50 nm). The area and diameter of each depressionopening or portion opening can be greater than, less than or between thevalues specified above. Any desirable areas and diameters may be used,as long as the area and diameter of the openings of depressions 28A arelarger than the area and diameter of the openings of depressions 28B, oras long as the area and diameter of the openings of portions 34 ofdepressions 28C are larger than the area and diameter of the openings ofportions 34′ of depressions 28C.

Many different layouts of the depressions 28A and 28B or 28C may beenvisaged, including regular, repeating, and non-regular patterns. In anexample, the depressions 28A and 28B or 28C are disposed in a hexagonalgrid for close packing and improved density. Other layouts may include,for example, rectilinear (i.e., rectangular) layouts, triangularlayouts, and so forth, as long as the depression 28A or the portion 34can receive a functionalized bead 50 (e.g., cores structure 49 withregion 16 formed thereon, as shown, e.g., in FIGS. 13C, 14, etc.). Insome examples, the layout or pattern can be an x-y format of depressions28A and 28B or 28C that are in rows and columns. In some other examples,the layout or pattern can be a repeating arrangement of depressions 28Aand 28B or 28C and/or interstitial regions 30. In still other examples,the layout or pattern can be a random arrangement of depressions 28A and28B or 28C and/or interstitial regions 30. The pattern may includestripes, swirls, lines, triangles, rectangles, circles, arcs, checks,plaids, diagonals, arrows, squares, and/or cross-hatches.

It is to be understood that the layout or pattern of the depressions 28Aand 28B or 28C may be characterized with respect to the density and/orthe average pitch as described herein.

Still further, the depressions 28A and 28B or 28C may have any suitabledepth.

An example of a method 100 for making an example of the flow cell usingthe patterned resin 54′ shown in FIG. 11C or the patterned resin 54″shown in FIG. 11D is depicted in FIG. 12. As shown in FIG. 12, themethod 100 includes selectively applying a polymer 32 in depressions28A, 28B or 28C of a patterned resin 54′ or 54″ on a support 52(reference numeral 102); grafting a first primer set 12A, 12B, 12C, 12Dto the polymer 32 in at least some of the depressions 28A, 28B or 28C(reference numeral 104); and before or after grafting the first primerset 12A, 12B, 12C, 12D, depositing functionalized beads 50 i) in aportion of each of the at least some of the depressions 28C, or ii) insecond depressions 28A having larger opening dimensions than the atleast some of the depressions 28B, the functionalized beads 50 includinga second primer set 12A′, 12B′, 12C′, 12D′, attached at a surface of acore structure 49, wherein the first and second primer sets 12A, 12A′ or12B, 12B′, or 12C, 12C′, or 12D, 12D′ are different. Different examplesof this method 100 involving the depressions 28A, 28B will be describedfurther in reference to FIGS. 13A through 13D and 14 and FIGS. 15Athrough 15D and 16. Different examples of this method 100 involving thedepressions 28C will be described further in reference to FIGS. 17Athrough 17D and 18 and FIGS. 19A through 19D and 20.

FIG. 13A depicts the patterned resin 54′ including larger depressions28A and smaller depressions 28B. The depth of the depressions 16A and16B is omitted for clarity.

FIG. 13B depicts the selective application of the polymer 32 into eachof the depressions 28A and 28B. The selective application of the polymer32 may involve multiple processes, including activation of theinterstitial regions 30 and the exposed surfaces in the depressions 28A,28B, depositing the polymer 32 on the activated interstitial regions 30and in the depressions 28A, 28B, and removing the polymer 32 from theinterstitial regions 30.

In some examples, activation involves silanizing the surface, includingthe interstitial regions 30 of the patterned resin 54′ and the regionsof the support 52 that are exposed in the depressions 28A, 28B.Silanization may be accomplished using any silane or silane derivative.The selection of the silane or silane derivative may depend, in part,upon the polymer 32 that is to be formed, as it may be desirable to forma covalent bond between the silane or silane derivative and the polymer32. The method used to attach the silane or silane derivative may varydepending upon the silane or silane derivative that is being used.Several examples are set forth herein.

Examples of suitable silanization methods include vapor deposition(e.g., a YES method), spin coating, or other deposition methods.

In an example utilizing the YES CVD oven, the support 52 having thepatterned resin 54′ thereon is placed in the CVD oven. The chamber maybe vented and then the silanization cycle started. During cycling, thesilane or silane derivative vessel may be maintained at a suitabletemperature (e.g., about 120° C. for norbornene silane), the silane orsilane derivative vapor lines be maintained at a suitable temperature(e.g., about 125° C. for norbornene silane), and the vacuum lines bemaintained at a suitable temperature (e.g., about 145° C.).

In another example, the silane or silane derivative (e.g., liquidnorbornene silane) may be deposited inside a glass vial and placedinside a glass vacuum desiccator with the support 52 having thepatterned resin 54′ thereon. The desiccator can then be evacuated to apressure ranging from about 15 mTorr to about 30 mTorr, and placedinside an oven at a temperature ranging from about 60° C. to about 125°C. Silanization is allowed to proceed, and then the desiccator isremoved from the oven, cooled and vented in air.

Vapor deposition, the YES method and/or the vacuum desiccator may beused with a variety of silane or silane derivatives, such as thosesilane or silane derivative including a cycloalkene unsaturated moiety,such as norbornene, a norbornene derivative (e.g., a (hetero)norborneneincluding an oxygen or nitrogen in place of one of the carbon atoms),transcyclooctene, transcyclooctene derivatives, transcyclopentene,transcycloheptene, trans-cyclononene, bicyclo[3.3.1]non-1-ene,bicyclo[4.3.1]dec-1 (9)-ene, bicyclo [4.2.1]non-1(8)-ene, andbicyclo[4.2.1]non-1-ene. Any of these cycloalkenes can be substituted,for example, with an R group, such as hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An example of thenorbornene derivative includes[(5-bicyclo[2.2.1]hept-2-enyl)ethyl]trimethoxysilane. As other examples,these methods may be used when the silane or silane derivative includesa cycloalkyne unsaturated moiety, such as cyclooctyne, a cyclooctynederivative, or bicyclononynes (e.g., bicyclo[6.1.0]non-4-yne orderivatives thereof, bicyclo[6.1.0]non-2-yne, orbicyclo[6.1.0]non-3-yne). These cycloalkynes can be substituted with anyof the R groups described herein.

The attachment of the silane or silane derivative forms an activatedsurface, both on the interstitial regions 30 and in the depressions 28A,28B.

The polymer layer 32 may then be applied as described herein. Asexamples, the polymer (e.g., PAZAM) may be deposited using spin coating,or dipping or dip coating, or flow of the functionalized molecule underpositive or negative pressure, or another suitable technique. Thepolymer deposited to form the polymer layer 32 may be present in amixture. In an example, the mixture includes PAZAM in water or in anethanol and water mixture.

After being coated, the mixture including the polymer may also beexposed to a curing process to form the polymer layer 32 across theactivated interstitial regions 30 of the patterned resin 54′ and in thedepressions 28A, 28B. In an example, curing may take place at atemperature ranging from room temperature (e.g., about 25° C.) to about95° C. for a time ranging from about 1 millisecond to about severaldays. In another example, the time may range from 10 seconds to at least24 hours. In still another example, the time may range from about 5minutes to about 2 hours.

The attachment of the polymer layer 32 to the activated (in this examplesilanized) surfaces may be through covalent bonding. Covalent linking ishelpful for maintaining at least the first primer set 12A, 12B, 12C, 12Din the depressions 28B throughout the lifetime of the ultimately formedflow cell during a variety of uses. The following are some examples ofreactions that can take place between the activated (e.g., silanized)surfaces and the polymer layer 32.

When the silane or silane derivative includes norbornene or a norbornenederivative as the unsaturated moiety, the norbornene or a norbornenederivative can: i) undergo a 1,3-dipolar cycloaddition reaction with anazide/azido group of PAZAM; ii) undergo a coupling reaction with atetrazine group attached to PAZAM; undergo a cycloaddition reaction witha hydrazone group attached to PAZAM; undergo a photo-click reaction witha tetrazole group attached to PAZAM; or undergo a cycloaddition with anitrile oxide group attached to PAZAM.

When the silane or silane derivative includes cyclooctyne or acyclooctyne derivative as the unsaturated moiety, the cyclooctyne orcyclooctyne derivative can: i) undergo a strain-promoted azide-alkyne1,3-cycloaddition (SPAAC) reaction with an azide/azido of PAZAM, or ii)undergo a strain-promoted alkyne-nitrile oxide cycloaddition reactionwith a nitrile oxide group attached to PAZAM.

When the silane or silane derivative includes a bicyclononyne as theunsaturated moiety, the bicyclononyne can undergo similar SPAAC alkynecycloaddition with azides or nitrile oxides attached to PAZAM due to thestrain in the bicyclic ring system.

In other examples, plasma ashing rather than silanization may be used toactivate the interstitial regions 30 and the exposed surfaces of thesupport 52 in the depressions 28A, 28B. After plasma ashing, the mixturecontaining the polymer may be directly spin coated (or otherwisedeposited) on the plasma ashed surfaces and then cured to form thepolymer layer 32. In this example, plasma ashing may generatesurface-activating agent(s) (e.g., hydroxyl (C—OH or Si—OH) and/orcarboxyl groups) that can adhere the polymer to the interstitial regions30 and the exposed surfaces of the support 52 in the depressions 28A,28B. In these examples, the polymer 26 is selected so that it reactswith the surface groups generated by plasma ashing.

Whether activation takes place via silanization or plasma ashing,polishing may then be performed in order to remove the polymer layer 32from the activated interstitial regions 30. Polishing may be performedas described herein. In some examples, polishing may or may not alsoremove the silane or silane derivative adjacent to the interstitialregions 30. When these silanized portions are completely removed, it isto be understood that the underlying patterned resin 54′ is exposed.

As shown in FIG. 13C, in this example of the method 100, functionalizedbeads 50 are deposited into the depressions 28A. Each functionalizedbead 50 includes a core structure 49 and the second primer set 12A′,12B′, 12C′, 12D′ attached at a surface of the core structure 49. Thesecond primer set 12A′, 12B′, 12C′, 12D′ is different than the firstprimer set 12A, 12B, 12C, 12D as described herein. Any examples of theprimer sets 12A, 12A′ or 12B, 12B′, or 12C, 12C′, or 12D, 12D′ may beused.

The core structure 49 may be any of the examples mentioned herein forthe bead/core structure. In an example, the core structure 49 of thefunctionalized bead 50 is selected from the group consisting of silicondioxide, a superparamagnetic material, polystyrene, and an acrylate. Thecore structure 49 may have reactive groups on its surface for covalentcoupling to the second primer set 12A′, 12B′, 12C′, 12D′. Examples ofsuch reactive groups include a carboxylic acid, a primary aliphaticamine, an aromatic amine, an aromatic chloromethyl (e.g., vinyl benzylchloride), an amide, a hydrazide, an aldehyde, a hydroxyl, a thiol, andan epoxy. These reactive group(s) may be inherently present at thesurface of the core structure 49, or may be incorporated on the surfaceof the core structure 49 through any suitable functionalizationtechnique (e.g., chemical reaction, coating the core structure 49 with areactive group-containing polymer, etc.). The core structure 49 with itsinherent reactive group(s) or its added reactive group(s) or coatingdefines the region 16.

In the examples disclosed herein that utilize the bead 50, one primerset 12A, 12B, 12C, 12D or 12A′, 12B′, 12C′, 12D′ could be functionalizedwith a phosphate blocking group at the 3′ end which would inhibit anyextension from occurring post seeding. Once the first extension occurs(with the other primer set 12A′, 12B′, 12C′, 12D′ or 12A, 12B, 12C,12D), the phosphate group would then be removed along with any strandsseeded to the phosphate blocked primer set 12A, 12B, 12C, 12D or 12A′,12B′, 12C′, 12D′ and amplification between the two primer groups 12A,12B, 12C, 12D and 12A′, 12B′, 12C′, 12D′ could then proceed. This methodwould help to reduce polyclonality rates since only one primer set 12A,12B, 12C, 12D or 12A′, 12B′, 12C′, 12D′ would be initially extendable.

While not shown in FIGS. 13A through 13D, prior to depositing thefunctionalized beads 50, the method 100 may include forming thefunctionalized beads 50 by attaching the second primer set 12A′, 12B′,12C′, 12D′ to the core structure 49. Functionalization may take placeusing any suitable technique, including reacting an azido (e.g.,succinimidyl (NHS) ester) terminated primer with a hydrazine on thesurface of the core structure 49, or reacting an alkyne terminatedprimer with an azide on the surface of the core structure, or reactingan amino terminated primer to an activated carboxylate group or NHSester on the surface of the core structure 49, or a thiol terminatedprimer with an alkylating reactant (e.g., iodoacetamine or maleimide), aphosphoramidite terminated primer with a thioether, or a biotin-modifiedprimer with streptavidin on the surface of the core structure 49. Somenucleic acid primers 19, 19′, 21, 21′ can be captured onto silica beadsin the presence of a chaotropic agent (KI, NI, or NaSCN). As onespecific example, a dibenzocyclooctyne (DBCO, which includes an alkyne)terminated primer may be used for copper free click grafting.

The functionalized beads 50 may be deposited onto the patterned resin54′ using any suitable technique. As one example, the functionalizedbeads 50 may be mixed in a liquid carrier (e.g., water), which can beloaded onto the surface of the patterned resin 54′. In another example,the functionalized beads 50 may be forcefully embedded into thedepressions 28A by shaking the beads 50 with 50 μm to 150 μm steel beadsin a shaker that also includes the support 52 having the patterned resin54′ thereon. Because of the size of the functionalized beads 50 and thesize of the depressions 28A and 28B, size exclusion will prevent thefunctionalized beads 50 from entering the smaller depressions 28B andwill enable one functionalized bead 50 to self-assemble into onedepression 28A. The functionalized beads 50 may be physically entrappedin the respective depressions 28A. The functionalized beads 50 mayalternatively be chemically attached in the respective depressions 28A,e.g., via streptavidin/biotin linkers.

As shown in FIG. 13D, the depressions 28B may then be functionalizedwith the first primer set 12A, 12B, 12C, 12D. The primers 18, 18′ and20, 20′ may be any of the examples disclosed herein, and includes afunctional group that can attach to a reactive group of the polymerlayer 32. Because the polymer layer 32 in the depressions 28A is coveredwith the functionalized beads 50, the polymer layer 32 in thedepressions 28A is not exposed, and thus its reactive groups are notavailable for reaction with the first primer set 12A, 12B, 12C, 12D.

The primers 18, 18′ and 20, 20′ may be any of the examples set forthherein, and are different from one another and different from primers19, 19′ and 21, 21′. For example, if primers 21, 19 include P5 and P7Uprimers, then primers 20, 18 may include the P5U and P7 primers. In thisexample, the first primer set 12A includes an un-cleavable first primer18 (e.g., P7) and a cleavable (e.g., uracil-modified) second primer(e.g., P5U); and the second primer set 12A′ includes a cleavable (e.g.,uracil-modified) first primer 19 (e.g., P7U) and an un-cleavable secondprimer 21 (e.g., P5). As discussed herein, the chemistry of the primersets 12A, 12A′ or 12B, 12B′ or 12C, 12C′ or 12D, 12D′ is orthogonal,which allows for amplification across both primer populations (sets 12A,12A′ or 12B, 12B′ or 12C, 12C′ or 12D, 12D′), and cleavage of some ofthe generated template strands (e.g., 42, 46), leaving the same (forwardor reverse) template strands 40 or 44 in a particular region 14 or 16.This enables distinguishable read 1 and read 2 signals to be obtainedsimultaneously.

A grafting process may be performed to graft the primers 18, 18′ and 20,20′ to the polymer layer 32 in the depressions 28B. In an example,grafting may be accomplished by flow through deposition (e.g., using atemporarily bound lid), dunk coating, spray coating, puddle dispensing,or by another suitable method that will attach the primer(s) 18, 18′ and20, 20′ to the polymer layer 32 in the depressions 28B. Each of theseexample techniques may utilize a primer solution or mixture, which mayinclude the primer(s), water, a buffer, and a catalyst.

Dunk coating may be used because the functionalized beads 50, in thisexample, are functionalized prior to being introduced into thedepressions 28A and thus are not reactive with the dunk chemistry. Dunkcoating may involve submerging the support 52 as shown in FIG. 13C (withpatterned resin 54′ thereon and functionalized beads 50 in depressions28A) into a series of temperature controlled baths. The baths may alsobe flow controlled and/or covered with a nitrogen blanket. The baths mayinclude the primer solution or mixture. Throughout the various baths,the primer(s) 18, 18′ and 20, 20′ attach to reactive group(s) of thepolymer layer 32. In an example, the support 52 will be introduced intoa first bath including the primer solution or mixture where a reactiontakes place to attach the primer(s) 18, 18′ and 20, 20′, and then movedto additional baths for washing. Movement from bath to bath may involvea robotic arm or may be performed manually. A drying system may also beused in dunk coating.

Spray coating may be accomplished by spraying the primer solution ormixture directly onto the support 52 as shown in FIG. 13C (withpatterned resin 54′ thereon and functionalized beads 50 in depressions28A). The spray coated wafer may be incubated for a time ranging fromabout 4 minutes to about 60 minutes at a temperature ranging from about0° C. to about 70° C. After incubation, the primer solution or mixturemay be diluted and removed using, for example, a spin coater.

Puddle dispensing may be performed according to a pool and spin offmethod, and thus may be accomplished with a spin coater. The primersolution or mixture may be applied (manually or via an automatedprocess) to the support 52 as shown in FIG. 13C (with patterned resin54′ thereon and functionalized beads 50 in depressions 28A). The appliedprimer solution or mixture may be applied to or spread across the entiresurface, including on the functionalized beads 50 and the interstitialregions 30. The primer coated substrate may be incubated for a timeranging from about 2 minutes to about 60 minutes at a temperatureranging from about 0° C. to about 80° C. After incubation, the primersolution or mixture may be diluted and removed using, for example, thespin coater.

With any of the grafting methods, the primers 18, 18′ and 20, 20′ reactwith reactive groups of the exposed polymer layer 32 in the depressions28B and have no affinity for the functionalized beads 50 or theinterstitial regions 30 of the patterned resin 54′. As such, the primers18, 18′ and 20, 20′ selectively graft to the polymer layer 32 in thedepressions 28B.

FIG. 14 is a cross-sectional view of the portion of the flow cell 10Adepicted in FIG. 13D. The flow cell 10A includes the support 52; thepatterned resin 54′ on the support 52, the patterned resin 54′ includingfirst depressions 28B and second depressions 28A separated byinterstitial regions 30, the first depressions 28B having smalleropening dimensions than the second depressions 28A; the first primer set12A, 12B, 12C, or 12D (including primers 18, 18′ and 20, 20′) attachedin at least some of the first depressions 28B; and the functionalizedbead 50 respectively positioned in at least some of the seconddepressions 28A, the functionalized bead 50 including a second primerset 12A′, 12B′, 12C′, or 12D′ (including primers 19, 19′ and 21, 21′)attached at a surface of a core structure 49, wherein the second primerset 12A′, 12B′, 12C′, or 12D′ is different than the first primer set12A, 12B, 12C, or 12D. In the example shown in FIGS. 13D and 14, thepolymer layer 32 is present in the first depressions 28B and in thesecond depressions 28A, and the first primer set 12A, 12B, 12C, or 12Dis attached to the polymer layer 32 in the at least some of the firstdepressions 28B. Moreover, in the example shown in FIGS. 13D and 14, thefunctionalized beads 50 are positioned on the polymer layer 32 in atleast some of the second depressions 28A.

Referring now to FIGS. 15A through 15D, another example of the method100 involving the depressions 28A, 28B is shown. FIG. 15A depicts thepatterned resin 54′ including larger depressions 28A and smallerdepressions 28B. The depth of the depressions 28A and 28B is omitted forclarity.

FIG. 15B depicts the polymer layer 32 in each of the depressions 28A and28B. The polymer layer 32 may be selectively applied as describedherein, which may include activating the interstitial regions 30 and theexposed surfaces in the depressions 28A, 28B, depositing the polymerlayer 32 on the activated interstitial regions 30 and in the depressions28A, 28B, and removing the polymer layer 32 from the interstitialregions 30.

In this example, as shown in FIG. 15C, the primer set 12A, 12B, 12C, 12D(including primers 18, 18′ and 20, 20′) is then grafted to the polymerlayer 32 in each of the depressions 28A, 28B. Because the functionalizedbeads 50 have not been introduced, the polymer layer 32 in each of thedepressions 28A, 28B is exposed, and thus its reactive groups areavailable for reaction with the first primer set 12A, 12B, 12C, 12D.Grafting may be accomplished using any of the techniques described inreference to FIG. 13D. Since the polymer layer 32 is exposed in each ofthe depressions 28A, 28B, the primers 18, 18′ and 20, 20′ can be graftedinto each of the depressions 28A, 28B.

As shown in FIG. 15D, the functionalized beads 50 may then be introducedinto the depressions 28A (having the polymer layer 32 and primer set12A, 12B, 12C, 12D therein). The functionalized beads 50 may bedeposited onto the patterned resin 54′ using any suitable technique.Because of the size of the functionalized beads 50 and the size of thedepressions 28A and 28B, size exclusion will prevent the functionalizedbeads 50 from entering the smaller depressions 28B and will enable onefunctionalized bead 50 to self-assemble into one depression 28A. In thisexample, the functionalized beads 50 may be physically entrapped in therespective depressions 28A. Alternatively in this example, thefunctionalized beads 50 may be chemically attached in the respectivedepressions 28A, e.g., via streptavidin/biotin linkers.

FIG. 16 is a cross-sectional view of the portion of the flow cell 10Bdepicted in FIG. 15D. The flow cell 10B includes the support 52; thepatterned resin 54′ on the support 52, the patterned resin 54′ includingfirst depressions 28B and second depressions 28A separated byinterstitial regions 30, the first depressions 28B having smalleropening dimensions than the second depressions 28A; the first primer set12A, 12B, 12C, 12D (including primers 18, 18′ and 20, 20′) attached inat least some of the first depressions 28B; and the functionalized bead50 respectively positioned in at least some of the second depressions28A, the functionalized bead 50 including a second primer set 12A′,12B′, 12C′, 12D′ (including primers 19, 19′ and 21, 21′) attached at asurface of a core structure 49, wherein the second primer set 12A′,12B′, 12C′, 12D′ is different than the first primer set 12A, 12B, 12C,12D. In the example shown in FIGS. 15D and 16, the polymer layer 32 ispresent in the first depressions 28B and in the second depressions 28A,and the first primer set 12A, 12B, 12C, 12D is attached to the polymerlayer 32 in the first depressions 28B and in the second depressions 28A.In this example then, the functionalized bead 50 is positioned on thefirst primer set 12A, 12B, 12C, 12D (including primers 18, 18′ and 20,20′) in the at least some of the second depressions 28A.

Referring now to FIGS. 17A through 17D, an example of the method 100involving the depressions 28C is shown.

FIG. 17A depicts the patterned resin 54″ including depressions 28C. Eachof the depressions 28C includes a larger portion 34 and a smallerportion 34′. The depth of the depressions 28C (as shown in FIG. 11D) isomitted for clarity.

FIG. 17B depicts the polymer layer 32 in each of the depressions 28C. Asshown in FIG. 17B, both the larger portion 34 and the smaller portion34′ of each depression 28C has the polymer layer 32 applied thereto. Thepolymer layer 32 may be selectively applied as described herein, which,in an example, may include activating the interstitial regions 30 andthe exposed surfaces in the depressions 28C, depositing the polymerlayer 32 on the activated interstitial regions 30 and in the depressions28C, and removing the polymer layer 32 from the interstitial regions 30.

As shown in FIG. 17C, in this example of the method 100, functionalizedbeads 50 are deposited into the larger portions 34 of the depressions28C. Any of the functionalized beads 50 described herein may be used inthis example.

The functionalized beads 50 (including primers 19, 19′ and 21, 21′) maybe deposited onto the patterned resin 54″ using any suitable technique,such as those described herein. Because of the size of thefunctionalized beads 50 and the size of the portions 34 and 34′, sizeexclusion will prevent the functionalized beads 50 from entering thesmaller portions 34′ and will enable one functionalized bead 50 toself-assemble into one larger portion 34 of each depression 28C. In thisexample, the functionalized beads 50 may be physically entrapped in therespective larger portions 34 or may be chemically attached in therespective larger portions 34, e.g., via streptavidin/biotin linkers.

As shown in FIG. 17D, the smaller portions 34′ of the depressions 28Cmay then be functionalized with the first primer set 12A, 12B, 12C, 12D.Any examples of the primers 18, 18′ and 20, 20′ described herein may beused, as long as they are selected to be different from and orthogonalto the primers 19, 19′ and 21, 21′ of the functionalized beads5. In thisexample, the polymer layer 32 in the larger portions 34 of thedepressions 28C is covered with the functionalized beads 50, and thusthe polymer layer 32 in the larger portions 34 of the depressions 28C isnot exposed. As such, the polymer reactive groups are not available forreaction with the first primer set 12A, 12B, 12C, 12D. The polymerreactive groups in the smaller portions 34′ remain exposed, and thus areavailable for reaction with the first primer set 12A, 12B, 12C, 12D.Grafting of the primers 18, 18′ and 20, 20′ to the polymer layer 32 inthe smaller portions 34′ may be accomplished using any of the techniquesdescribed in reference to FIG. 13D.

FIG. 18 is a cross-sectional view of the portion of the flow cell 10Cdepicted in FIG. 17D. The flow cell 10C includes the support 52; thepatterned resin 54″ on the support 52, the patterned resin 54″ includingdepressions 28C separated by interstitial regions 30; the first primerset 12A, 12B, 12C, 12D (including primers 18, 18′ and 20, 20′) attachedto at least some of the depressions 28C; and the functionalized bead 50positioned in the at least some of the depressions 28C so that at leastsome primers 18, 18′ and 20, 20′ of the first primer set 12A, 12B, 12C,12D are exposed, the functionalized bead 50 including the second primerset 12A′, 12B′, 12C′, 12D′ attached at a surface of the core structure49, wherein the second primer set 12A′, 12B′, 12C′, 12D′ is differentthan the first primer set 12A, 12B, 12C, 12D. In the example shown inFIGS. 17D and 18, each of the depressions 28C includes a first portion34 with a first opening dimension that is larger than or equal to adiameter of the functionalized bead 50, and a second portion 34′ with asecond opening dimension that is smaller than the diameter of thefunctionalized bead 50; and the functionalized bead 50 is positioned inthe first portion 34 of each of the at least some of the depressions28C. Also in the example shown in FIGS. 17D and 18, the polymer layer 32is present in the depressions 28C, and the first primer set 12A, 12B,12C, 12D is attached to a portion of the polymer layer 32 unoccupied bythe functionalized bead 50 (i.e., to the portion of the polymer layer 32in the smaller portion 34′ of the depression 28C).

Referring now to FIGS. 19A through 19D, another example of the method100 involving the depressions 28C is shown. Each of the depressions 28Cincludes a larger portion 34 and a smaller portion 34′. The depth of thedepressions 28C (as shown in FIG. 11D) is omitted for clarity.

FIG. 19B depicts the polymer layer 32 in each of the depressions 28C. Asshown in FIG. 19B, both the larger portion 34 and the smaller portion34′ of each depression 28C has the polymer layer 32 applied thereto. Thepolymer layer 32 may be selectively applied as described herein, which,in this example, may include activating the interstitial regions 30 andthe exposed surfaces in the depressions 28C, depositing the polymerlayer 32 on the activated interstitial regions 30 and in the depressions28C, and removing the polymer layer 32 from the interstitial regions 30.

In this example, as shown in FIG. 19C, the primer set 12A, 12B, 12C, 12D(including primers 18, 18′ and 20, 20′) is then grafted to the polymerlayer 32 in each of the depressions 28C. Because the functionalizedbeads 50 have not been introduced, the polymer layer 32 in each portion34, 34′ of the depressions 28C is exposed, and thus its reactive groupsare available for reaction with the first primer set 12A, 12B, 12C, 12D.Grafting may be accomplished using any of the techniques described inreference to FIG. 13D. Since the polymer layer 32 is exposed in eachportion 34, 34′ of the depressions 28C, the primers 18, 18′ and 20, 20′can be grafted into each of the each portion 34, 34′.

As shown in FIG. 19D, the functionalized beads 50 may then be introducedinto the larger portions 34 of the depressions 28C (having the polymerlayer 32 and primer set 12A, 12B, 12C, 12D therein). The functionalizedbeads 50 may be deposited onto the patterned resin 54″ using anysuitable technique. Because of the size of the functionalized beads 50and the size of the portions 34 and 34′, size exclusion will prevent thefunctionalized beads 50 from entering the smaller portions 34′ and willenable one functionalized bead 50 to self-assemble into one largerportion 34. In this example, the functionalized beads 50 may bephysically entrapped in the respective larger portions 34, or may bechemically attached in the respective larger portions 34, e.g., viastreptavidin/biotin linkers.

FIG. 20 is a cross-sectional view of the portion of the flow cell 10Ddepicted in FIG. 19D. The flow cell 10D includes the support 52; thepatterned resin 54″ on the support 52, the patterned resin 54″ includingdepressions 28C separated by interstitial regions 30; the first primerset 12A, 12B, 12C, 12D (including primers 18, 18′ and 20, 20′) attachedto at least some of the depressions 28C; and the functionalized bead 50positioned in the at least some of the depressions 28C so that at leastsome primers 18, 18′ and 20, 20′ of the first primer set 12A, 12B, 12C,12D are exposed, the functionalized bead 50 including the second primerset 12A′, 12B′, 12C′, 12D′ attached at a surface of the core structure49, wherein the second primer set 12A′, 12B′, 12C′, 12D′ is differentthan the first primer set 12A, 12B, 12C, 12D. In the example shown inFIGS. 19D and 20, each of the depressions 28C includes a first portion34 with a first opening dimension that is larger than or equal to adiameter of the functionalized bead 50, and a second portion 34′ with asecond opening dimension that is smaller than the diameter of thefunctionalized bead 50; and the functionalized bead 50 is positioned inthe first portion 34 of each of the at least some of the depressions28C. Also in the example shown in FIGS. 19D and 20, the polymer layer 32is present in the depressions 28C, the first primer set 12A, 12B, 12C,12D is attached to the polymer layer 32 in the depressions 28C, and thefunctionalized bead 50 is positioned on some of the primers 18, 18′ and20, 20′ of the first primer set 12A, 12B, 12C, 12D (i.e., on the primers18, 18′ and 20, 20′ attached to the polymer layer 32 in the largerportion 34 of the depression 28C).

The example flow cells 10A, 10B, 10C, 10D are shown without a lid bondedthereto. While not shown, the flow cells 10A, 10B, 10C, 10D may have thelid bonded to at least a portion of the interstitial region 30 asdescribed. In some examples, the lid may be bonded after the flow cell10A, 10B, 10C, 10D is formed.

The flow cells 10A, 10B, 10C, 10D may be used in a variety of sequencingapproaches or technologies, including techniques often referred to assequencing-by-synthesis (SBS), cyclic-array sequencing,sequencing-by-ligation, pyrosequencing, and so forth. In one specificexample, flow cells 10A, 10B, 10C, 10D may be exposed to the templatefluid/mix, and amplification may be performed as described herein togenerate the un-cleavable first template strand 40, the cleavable firsttemplate strand 46, the un-cleavable second template strand 44, thecleavable second template strand 42. Cleavage may be performed, and thenthe simultaneously paired-end sequencing method disclosed herein may beperformed.

With any of the techniques used with the flow cells 10A, 10B, 10C, 10D,since the primer sets 12A, 12A′ or 12B, 12B′ or 12C, 12C′, or 12D, 12D′are present in the depressions 28A or 28B or portions 34 or 34′ ofdepression 28C and not on the interstitial regions 30, amplification andsequencing will be confined to the depressions 28A and 28B or 28C.

Block-Copolymer Based Flow Cell

Some examples of the flow cell disclosed herein include the blockcopolymer, which is used with the multi-layered substrate including apatterned resin. These examples are described in reference to FIG. 21through FIG. 25B.

In these examples, the patterned resin (e.g., 54′) can be fabricatedusing a “top down” approach, such as nanoimprint lithography. Top downapproaches can generate an array of depressions 28 with a high density,a low pitch, and small nanofeatures. When combined with the directedself-assembly of the block copolymer, which is a “bottom up” approach,even smaller features (having sub-lithographic size domains) may beformed within the depressions 28. Small features may be desirablebecause a higher cluster density may be obtained. Higher cluster densitymeans that more bases can be read from a given unit area, whichincreases the genetic yield from the patterned flow cell. Moreover, thenon-grafted regions (e.g., interstitial regions 30) surround the smallfeatures, which enable greater accessibility to primers grafted to theblock copolymer. As such, utilization of the primers may increase.

In these examples, the block copolymer is self-assembled in thedepressions 28 of the patterned resin, and not to interstitial regions30 between the depressions 28. As such, additional processing forremoval of material from the interstitial regions 30 is not involved.

These examples of the flow cell include a support 52; a patterned resinon the support 52, the patterned resin including depressions 28separated by interstitial regions 30; a block copolymer on the patternedresin in the depressions 28, each block of the block copolymer having ablock-specific functional group that is different from theblock-specific functional group of each other block of the blockcopolymer; and a primer attached to the block-specific functional groupof at least one of the blocks. In some examples, the primers includeun-cleavable primers 18, 18′ and 21, 21′ attached to one block of theblock-copolymer, and the flow cell may be used in paired-end sequencingtechniques that involve sequentially sequencing forward template strandsand then reverse template strands that are attached to the one block. Inother examples, the respective primer sets 12A, 12A′, or 12B, 12B′, or12C, 12C′, or 12D, 12D′ disclosed herein are attached to differentblocks of the block-copolymer. In this example, one block includesprimers 18, 18′ and 20, 20′ and another block includes primers 19, 19′and 21, 21′. This example of the flow cell may be used in thesimultaneous paired-end sequencing techniques disclosed herein.

Examples of portions of the flow cells are shown in FIGS. 22E and 23,and will be described further herein.

An example of a method 200 for making an example of the flow cell isshown in FIG. 21. As shown in FIG. 21, the method 100 includespatterning a resin to form a patterned resin including depressions 28separated by interstitial regions 30 (reference numeral 202);introducing a solution including a block copolymer on the patternedresin, each block of the block copolymer having a block-specificfunctional group that is different from the block-specific functionalgroup of each other block of the block copolymer (reference numeral204); exposing the solution to solvent vapor annealing, whereby theblock copolymer phase separates and self-assembles in the depressions 28(reference numeral 206); and grafting a primer to the block-specificfunctional group of at least one of the blocks (reference numeral 208).This method 200 will be described further in reference to FIGS. 22Athrough 22E.

FIG. 22A depicts the support 52, and FIG. 22B depicts a resin 54Adeposited on the support 52. Any examples of the support 52 describedherein may be used. With the block copolymer, the resin 54A is capableof having depressions 28 (FIG. 22C) defined therein and is also capableof acting as a guiding template for the subsequently deposited blockcopolymer 58 (“BCP” between FIGS. 22C and 22D). As such, any resin 54Athat can be patterned using photolithography, nanoimprint lithography,stamping techniques, embossing techniques, molding techniques,microetching techniques, printing techniques, etc. may be used.Moreover, the resin 54A, after being patterned (i.e., patterned resin54A′), should have a surface energy that is within the same range of theblock copolymer 58 that is to be deposited thereon. In an example, theresin 54A/patterned resin 54A′ and the block copolymer 58 each have asurface energy within a range of from about 25 mN/m to about 50 mM/m.

Some examples of resins 54A that can be patterned and act as guidingtemplates are selected from the group consisting of a polyhedraloligomeric silsesquioxane resin (POSS)-based resin, an epoxy resin, apoly(ethylene glycol) resin, a polyether resin, an acrylic resin, anacrylate resin, a methacrylate resin, and combinations thereof. Whileseveral examples have been provided, it is believed that any resin thatcan be radical cured may be used. Any of the resins 54 disclosed hereinmay be used for the resin 54A.

As shown between FIG. 22A and FIG. 22B, the resin 54A is deposited onthe support 52. In an example of the method 200, deposition of the resin44 involves chemical vapor deposition, dip coating, dunk coating, spincoating, spray coating, puddle dispensing, ultrasonic spray coating,doctor blade coating, aerosol printing, screen printing, microcontactprinting, or inkjet printing.

The deposited resin 54 is then patterned, using any of the patterningtechniques mentioned herein. In the example shown in and between FIG.22A and FIG. 22B, nanoimprint lithography is used to pattern the resin54. After the resin 54 is deposited, it may be soft baked to removeexcess solvent. A nanoimprint lithography mold or working stamp 56 ispressed against the layer of resin 54 to create an imprint on the resin54. In other words, the resin 54 is indented or perforated by theprotrusions of the working stamp 56. The resin 54 may be then be curedwith the working stamp 56 in place. Curing may be accomplished byexposure to actinic radiation, such as visible light radiation orultraviolet (UV) radiation, or to radiation of a wavelength ranging fromabout 240 nm and 380 nm when a photoresist is used; or by exposure toheat when a thermal-curable resist is used. Curing may promotepolymerization and/or cross-linking. As an example, curing may includemultiple stages, including a softbake (e.g., to drive off solvent(s))and a hardbake. The softbake may take place at a lower temperature,ranging from about 50° C. to about 150° C. The duration of the hardbakemay last from about 5 seconds to about 10 minutes at a temperatureranging from about 100° C. to about 300° C. Examples of devices that canbe used for softbaking and/or hardbaking include a hot plate, oven, etc.

After curing, the working stamp 56 is released. This creates topographicfeatures, i.e., the depressions 16, in the resin 54. As shown in FIG.22C, the resin 54 having the depressions 28 defined therein is referredto as the patterned resin 54′. The patterned resin 54′ may be subject tofurther hard baking to complete the cure and to lock in the imprintedtopography. In some examples, the hard baking may be performed at atemperature ranging from about 60° C. to about 300° C.

As shown in FIG. 22C, the patterned resin 54′ includes the depressions28 defined therein, and interstitial regions 30 separating adjacentdepressions 28. In the examples disclosed herein, the depressions 28become functionalized with the block copolymer (BCP) 58 and primers 18,21 (FIG. 22F), while portions of the interstitial regions 20 may be usedfor bonding but will not have the block copolymer 58 or the primer(s)18, 21.

As shown in FIG. 22C, the patterned resin 54′ may then be exposed toprocesses to form the phase separated block copolymer 58 (includingblocks 58A and 58B) in the depressions 28. As shown between FIG. 22C andFIG. 22D, a solution of the block copolymer 58 is deposited on thepatterned resin 54′, where each block 58A, 58B of the block copolymer 58has a block-specific functional group that is different from theblock-specific functional group of each other block 58B, 58A of theblock copolymer 58. Various examples of the block copolymer 58 will nowbe described.

The block copolymer 58 is a heteropolymer made up of at least twodifferent monomers. In one example, block copolymer 58 includes a firstblock 58A including a monomer having a primer-grafting functional groupas its block-specific functional group, and a second block 58B includinga monomer that is to adjust an interaction parameter to drive phaseseparation of the first and second blocks. In this example, the monomerof the second block 58B may also include a block-specific functionalgroup that can react with (and thus attach to) the patterned resin 54′.The block-specific functional group that can react with (and thus attachto) the patterned resin 54′ is referred to herein as the resin-attachingfunctional group. It is to be understood that the designations first andsecond do not indicate any particular order in the block copolymer, andthat any block may include a primer-grafting functional group or afunctional group to adjust an interaction parameter. For an example, thefirst block 58A may include a monomer having a block-specific functionalgroup that is able to graft a primer (e.g., 18, 21) and that is able toadjust an interaction parameter to drive phase separation of the firstand second blocks, and the second block 58B may include a monomer havingthe resin-attaching functional group. For another example, the firstblock 58A may include a monomer having a block-specific functional groupthat able to graft a primer (e.g., 18, 21) and attach to the resin 54′,and that the second block 58B may include a monomer that is able toadjust an interaction parameter to drive phase separation of the firstand second blocks.

In any examples of the block copolymer 58 disclosed herein, theprimer-grafting functional group is selected from the group consistingof azide/azido, optionally substituted amino, optionally substitutedalkenyl, aldehyde, optionally substituted hydrazone, optionallysubstituted hydrazine, carboxyl, hydroxy, optionally substitutedtetrazole, optionally substituted tetrazine, nitrile oxide, nitrone,thiol, and combinations thereof. When multiple primer-graftingfunctional groups are included in a single block, different primers maybe attached to the single block. When different primer-graftingfunctional groups are included in different blocks, different primersmay be attached to the different blocks.

The primer-grafting functional group may be capable of reacting with afunctional group attached to the 5′ end of the primer. For example, abicyclo[6.1.0] non-4-yne (BCN) terminated primer may be captured by anazide primer-grafting functional group of the block copolymer 58 viastrain-promoted catalyst free click chemistry. For another example, analkyne terminated primer may be captured by an azide primer-graftingfunctional group of the block copolymer 58 via copper catalyzed clickchemistry. For still another example, a norbornene terminated primer mayundergo a catalyst-free ring strain promoted click reaction with atetrazine primer-grafting functional group of the block copolymer 58.

In an example, the primer-grafting functional group is an azido groupattached to an acrylamide monomer. An example of this monomer is azidoacetamido pentyl acrylamide. In another example, the primer-graftingfunctional group is an azido group attached to a benzene-containingmonomer. Two examples of this monomer include benzyl azide or an azidefunctionalized styrene

In any examples of the block copolymer 58 disclosed herein, theresin-attaching functional group is selected from the group consistingof an amino group, an alcohol group, an aryl group, and a charged group.Suitable anionic charged groups include sulfates or carboxylic acids.Suitable cationic charged groups include ammonium, guanidinium, orimidazolium. In other examples, the resin-attaching functional group maya trifluoromethyl group (—CF₃). In still another example of the blockcopolymer 18 disclosed herein, the monomer including the resin-attachingfunctional group may be a siloxane monomer, such as SiO(CH₃)₂.

In an example, the resin-attaching functional group is an amino groupattached to an acrylamide monomer. An example of this monomer is

In this example, the ethyl bridge (between the nitrogens) may bereplaced with a propyl bridge or any other bridge length that does notinterfere with the desired function of the monomer. In an example, thebridge length may be up to 16 carbon atoms. In another example, theresin-attaching functional group is an aryl group of a styrene monomer.Other resin attaching groups (for covalent attachment) depend on how theresin 14 is functionalized. For example, if the resin includes epoxygroups, an amine or alcohol may be a suitable resin-attaching functionalgroup.

Some examples of the block copolymer 58 include the primer-graftingfunctional group and the resin-attaching functional group. These groupsmay depend, respectively and in part, upon the primer (e.g., 18, 21 or18, 20 or 18′, 20′ or 19, 21 or 19′, 21′) to be grafted and upon thepatterned resin 54′ that is to attach to the block copolymer 58. Thefollowing are some examples of block copolymers 58 that include both theprimer-grafting functional group and the resin-attaching functionalgroup.

In an example where the patterned resin 54′ is an epoxy POSS, the blockcopolymer 58 includes a first block 58A including an acrylamide monomerhaving an amino group as its block-specific functional group, and asecond block 58B including an azido acetamido pentyl acrylamide monomerhaving an azido group as its block-specific functional group. In thisexample, the first block 58A includes the resin-attaching functionalgroup and the second block 58B includes the primer-grafting functionalgroup, although the second block 58B may also function as aresin-attaching functional group. A specific example of this blockcopolymer 18 is:

wherein R is hydrogen or a polymer initiating species end group, nranges from 1 to 10,000, and m ranges from 1 to 10,000. Examples of thepolymer initiating species end group include a reversibleaddition-fragmentation chain transfer (RAFT) end group, an atom transferradical polymerization (ATRP) end group, a nitroxide-mediated radicalpolymerization (NMP) end group, a tetramethylethylenediamine (TEMED) endgroup, or a free-radical polymerization (FRP) end group. In anotherexample, n and m independently range from about 1 to about 1,000.

In another example where the patterned resin 54′ is an epoxy POSS, theblock copolymer 58 includes a first block 58A including a styrenemonomer having an aryl group as its block-specific functional group, anda second block 58B including an azide functionalized styrene having anazido as its block-specific functional group. In this example, the firstblock 58A includes the resin-attaching functional group and the secondblock 58B includes the primer-grafting functional group, although thesecond block 58B may also function as a resin-attaching functionalgroup. A specific example of this block copolymer 58 is:

wherein n ranges from 1 to 10,000, and m ranges from 1 to 10,000. Inanother example, n and m independently range from about 1 to about1,000.

In still another example where the patterned resin 54′ is an epoxy POSS,the block copolymer 58 includes a first block 58A including an azidefunctionalized styrene having an azido as its block-specific functionalgroup, and a second block 58B including a siloxane monomer having thesiloxane as its block-specific functional group. In this example, thefirst block 58A includes the primer-grafting functional group, althoughthe first block 58A may also function as a resin-attaching functionalgroup, and the second block 58B includes the resin-attaching functionalgroup. A specific example of this block copolymer 58 is:

wherein n ranges from 1 to 10,000, and m ranges from 1 to 10,000. Inanother example, n and m independently range from about 1 to about1,000.

In an example where the patterned resin 54′ is an amorphousfluoropolymer (such as CYTOP®), the block copolymer 58 includes a firstblock 58A including a monomer having a trifluoromethyl group as itsblock-specific functional group, and a second block 58B including amonomer having a primer-grafting and resin-grafting functional group asits block-specific functional group. In one example of this blockcopolymer 58, the second block 58B includes the azido acetamido pentylacrylamide monomer and the first block 58A (which, in this example, maybe a surface energy altering functional group) may be a fluorinatedacrylate or a fluorinated acrylamide. Specific examples of this blockcopolymer 18 have the structure:

wherein n ranges from 1 to 10,000, and m ranges from 1 to 10,000. Inanother example, n and m independently range from about 1 to about1,000. In another example of this block copolymer 58 (which is suitablefor use with an amorphous fluoropolymer), the second block 58B includesazide functionalized styrene and the first block 58A (in this examplethe surface energy altering functional group) may be trifluoroethylacrylate. A specific example of this block copolymer 58 has thestructure:

wherein n ranges from 1 to 10,000, and m ranges from 1 to 10,000. Inanother example, n and m independently range from about 1 to about1,000.

It is to be further understood that the block copolymers 58 disclosedherein may also include one or more other monomers that do not interferewith the respective functions of the blocks 58A and/or 58B (e.g., primergrafting, resin attaching, phase separating, etc.). The additionalmonomer(s) (and specifically the block-specific functional group of theadditional monomer(s)) of the additional block(s) may be selected toaffect/alter a surface free energy of the block copolymer 58, to affectthe stability of the block copolymer 58, to attach another primer,and/or to attach an enzyme. Examples of monomers that can affect/alterthe surface free energy include a trifluoromethyl group of an acrylatemonomer (e.g., trifluoroethyl acrylate or trifluoroethyl methacrylate)or of trifluoroethyl acrylamide. Examples of monomers that can attach anenzyme may include the following block-specific functional groups:thiols, amines, or alcohols, which can react with N-hydroxysuccinimide(NHS)-functionalized enzymes. It is to be understood that otherfunctional groups may be used to attach enzymes or other biomolecules.As such, some examples of the block copolymer 58 are terpolymers, whichwill be discussed in further detail in reference to FIG. 24A and FIG.24B.

As mentioned above, in the method 200, a solution of the block copolymer58 is introduced on the patterned resin 54′ (as shown between FIGS. 22Cand 22D). The solution may be a dilute solution (e.g., ranging fromabout 0.01 wt % to about 10 wt %) of the block copolymer 58 in asuitable solvent, such as toluene. The block copolymer 58 solution maybe deposited using any suitable technique, such as spin coating, etc.

For the block copolymer 58 to self-assemble and undergo microphaseseparation on the topologically patterned support (e.g., patterned resin54′), the solution including the block copolymer is to have a highHuggins interaction parameter with the underlying patterned resin 54′.In an example, the solution of the block copolymer 58 has aFlory-Huggins interaction parameter ranging from about 0.04 to about0.30. In another example, the solution of the block copolymer 58 has aFlory-Huggins interaction parameter of about 0.26.

The as-deposited block copolymer 58 on the patterned resin 54′ is thenexposed to solvent annealing. The solvent vapor, temperature, and timeused in solvent annealing may depend upon the block copolymer 58 used,and, in particular, on the conditions at which the block copolymer 58self-assembles into the depressions 28 and microphase separates into therespective blocks 58A and 58B. In an example, the solvent vapor istoluene, the temperature is room temperature (e.g., from about 14° C. toabout 25° C.), and the time is about 3 hours. It is to be understoodthat the solvent selection, annealing time, and annealing temperaturedepend on the block copolymer. Some suitable solvents may be toluene,heptane, higher alkanes and mixtures thereof. The time and temperaturemay influence the morphology in the depressions 28, and thus may becontrolled. As example, annealing time may range from 1 minute to 180minutes, or even longer, for example, from about 3 hours to about 48hours; and the annealing temperature may range from 18° C. to about 250°C.

As a result of solvent annealing, the block copolymer 58 self-assemblesinto the depressions 16 (and thus is not present on the interstitialregions 30) and also phase separates into segregated domains, or blocks58A, 58B. In FIG. 2D, the segregated blocks 58A, 58B have a circular orspiral pattern, although the pattern may depend upon the block copolymer58 used. Other examples of the pattern of the block copolymer 58 areshown, for example, in FIGS. 23A and 23B. FIG. 23A is a top view of adepression 28 and some of the surrounding interstitial region 30, wherethe block copolymer 58 in the depression 28 phase separates into blocks58A and 58B that exhibit a fingerprint pattern. FIG. 23B is a top viewof a depression 28 and some of the surrounding interstitial region 30,where the block copolymer 58 in the depression 28 phase separates intoblocks 58A and 58B that exhibit a line pattern. The blocks 58A, 58B havesub-lithographic size domains.

During solvent annealing the block 58A or 58B including theresin-attaching functional group may react with the patterned resin 54′,and thus may attach to the patterned resin 54′.

While not shown, in some examples of the method 200, the patterned resin54′, including the phase separated and self-assembled block copolymer58A, 58B in the depressions 28 is exposed to an additional curingprocess. Curing may be performed as previously described.

As shown between FIGS. 22D and 22E, a grafting process is performed inorder to graft primers (18, 21 or 18′, 21′ for sequential paired-endsequencing) or (18, 20 or 18′, 20′ and 19, 21 or 19′, 21′ forsimultaneous paired-end sequencing) to any primer-grafting functionalgroups of the block(s) 58A and/or 58(B) in the depression(s) 28.

In an example, grafting may be accomplished by flow through deposition(e.g., using a temporarily bound lid), dunk coating, spray coating,puddle dispensing, or by another suitable method that will attach theprimer(s) (18, 21 or 18′, 21′ for sequential paired-end sequencing) or(18, 20 or 18′, 20′ and 19, 21 or 19′, 21′ for simultaneous paired-endsequencing) to the primer-grafting functional groups of the block(s) 58Aand/or 58B. In an example of simultaneous paired-end sequencing, primers18, 20 or 18′, 20′ may be grafted to one block 58A and primers 19, 21 or19′, 21′ may be grafted to the other block 58B. Each of these exampletechniques may be performed as described herein and may utilize a primersolution or mixture, which may include the primer(s), water, a buffer,and a catalyst.

It is to be understood that primer(s) (un-cleavable primers 18, 21 or18′, 21′ for sequential paired-end sequencing) or(un-cleavable/cleavable primer pairs 18, 20 or 18′, 20′ and 19, 21 or19′, 21′ for simultaneous paired-end sequencing) will attach to theblock(s) 58A and/or 58(B) that include the primer-grafting functionalgroups. In the example shown in FIGS. 22E and 22F, the primer(s) 18, 21are attached to the block 58B and not to the block 58A. In this example,block 58A may have contributed the interaction parameter to drive phaseseparation of the first and second blocks 58A, 58B, and may also beattached to the patterned resin 54′ through a resin-attaching functionalgroup.

Referring now to FIG. 24, another example is depicted with the blockcopolymer 58 phase separated into two blocks 58A and 58B. In thisexample, two different primer sets, one including primers 18, 18′ and20, 20′ and the other including primers 19, 19′ and 21, 21′ are attachedto the respective blocks 58A, 58B. This example enables simultaneouspaired end reads during sequencing as described herein. In this example,each of the respective blocks 58A, 58B includes primer-graftingfunctional groups that can attach the respective primer sets.

As mentioned above, some of the block copolymers 58 are terpolymers,where each block includes a different block-specific functional group.In one example, the block copolymer 58 is a terpolymer including a firstblock, a second block, and a third block; where the block-specificfunctional group of the first block is attached to the patterned resin(i.e., is the resin-attaching functional group); the block-specificfunctional group of the second block is attached to primer(s) (i.e., isthe primer-grafting functional group); and the block-specific functionalgroup of the third block is attached to i) another primer(s) that isdifferent than the primer(s) attached to the block-specific functionalgroup of the second block or ii) to an enzyme (e.g., NEXTERA™transposomes). An example of the phase separated terpolymer is shown inFIG. 25A. In this example, the segregated terpolymer includes blocks58A, 58B, 58C, where 58A includes the primer-grafting functional group,58B includes the resin-attaching functional group and/or affects asurface free energy of the block copolymer and/or affects stability ofthe block copolymer, and 58C includes a different primer-graftingfunctional group or an enzyme attaching functional group. As depicted,block 58A attaches the first primer set 12A, 12B, 12C, 12D, includingprimers 18, 18′ and 20, 20′, and block 58C attaches the second primerset 12A′, 12B′, 12C′, 12D′, including primers 19, 19′ and 21, 21′. Inthis example, block 58A serves as region 14 and block 58C serves asregion 16. In another example, the regions 14, 16 may be blocks (e.g.,58A and 58B or 58B and 58C) that are directly adjacent to one another.

In another example, the block copolymer 58 is a terpolymer including afirst block, a second block, and a third block; where the block-specificfunctional group of the first block is attached to the patterned resin54′ (i.e., is the resin-attaching functional group); the block-specificfunctional group of the second block is attached to the primer(s) (i.e.,is the primer-grafting functional group); and the block-specificfunctional group of the third block affects a surface free energy of theblock copolymer or affects stability of the block copolymer. An exampleof the phase separated terpolymer is shown in FIG. 25B. In this example,the segregated terpolymer includes blocks 58A, 58B, 58C, where 58Aincludes the resin-attaching functional group, 58B affects the surfacefree energy of the block copolymer or affects stability of the blockcopolymer, and 58C includes a primer-grafting functional group. In thisexample, un-cleavable primers 18, 21 are attached, and thus this examplemay be particularly suitable for sequential paired-end sequencing.

FIG. 38A and FIG. 38B together depict another example method involving ablock copolymer 58′. In this example, the block copolymer 58′ is alamellar block copolymer. A lamellar block copolymer will self-assembleso that the different blocks 58A′ and 58B′ are layered one on top of theother so that they are parallel to the underlying materials 54B, 54C,and support 52. Examples of lamellar block copolymers include copolymersincluding heterocyclic azide units. The materials 54B, 54C may bedifferent examples of the resin 54A described herein, silanes, orsilanized resins, and may be selected to guide the self-assembly of theblock copolymer 58′. Under controlled conditions, the block copolymer58′ self-assembles into specific domains/blocks 58A′ and 58B′ that arelayered on the underlying materials 54B, 54C. In the example shown inFIG. 38A and 38B, as a result of annealing, the domains/blocks 58A′ and58B′ self-assemble so that block 58′B is exposed at the surface at onearea (overlying material 54B) and so that the other block 58A′ isexposed at the surface at another area (overlying material 54C). Theblock 58B′ may include primer-attaching functional groups that canattach the first primer set 12A, 12B, 12C, 12D, including primers 18,18′ and 20, 20′; and the block 58A′ may include differentprimer-attaching functional groups that can attach the second primer set12A′, 12B′, 12C′, 12D′, including primers 19, 19′ and 21, 21′. In thisexample, block 58B′ serves as region 14 and block 58A′ serves as region16.

The flow cells including the block copolymer 58, 58′ may be used in avariety of sequencing approaches or technologies, including techniquesoften referred to as sequencing-by-synthesis (SBS), cyclic-arraysequencing, sequencing-by-ligation, pyrosequencing, and so forth. Insome of these examples, since the phase separated blocks 58A, 58B, 58Cand attached primer(s) (un-cleavable primers 18, 21 or 18′, 21′ forsequential paired-end sequencing) or (un-cleavable/cleavable primerpairs 18, 20 or 18′, 20′ and 19, 21 or 19′, 21′ for simultaneouspaired-end sequencing) are present in the depressions 28 and not on theinterstitial regions 30, amplification will be confined to thedepressions 28.

As one example, a sequencing by synthesis (SBS) reaction may be run on asystem such as the HISEQ™, HISEQX™, MISEQ™, MISEQDX™, MINISEQ™,NOVASEQ™, NEXTSEQDX™, NEXTSEQ™, or any other sequencer systems fromIllumina (San Diego, Calif.). In SBS, extension of a sequencing primeralong a nucleic acid template (e.g., the sequencing template) ismonitored to determine the sequence of nucleotides in the template. Theunderlying chemical process can be polymerization (e.g., catalyzed by apolymerase enzyme) or ligation (e.g., catalyzed by a ligase enzyme). Ina particular polymerase-based SBS process, fluorescently labelednucleotides are added to the sequencing primer (thereby extending theprimer) in a template dependent fashion such that detection of the orderand type of nucleotides added to the sequencing primer can be used todetermine the sequence of the template.

For example, to initiate a first SBS cycle, one or more labelednucleotides, DNA polymerase, etc., may be delivered into/through theflow channel, etc. that houses an array of forward or reverse strandsattached to un-cleavable primers 18, 21 or 18′, 21′ (for sequentialpaired-end sequencing), or both forward and reverse strands attached tothe un-cleavable/cleavable primer pairs 18, 20 or 18′, 20′ (forsimultaneous paired-end sequencing), where sequencing primer extensioncauses a labeled nucleotide to be incorporated, can be detected throughan imaging event. During an imaging event, an illumination system (notshown) may provide an excitation light to the functionalizeddepressions.

In these example, the nucleotides can further include a reversibletermination property that terminates further primer extension once anucleotide has been added, and a deblocking agent can be used tocontinue sequencing. Wash(es) may take place between the various fluiddelivery steps. The SBS cycle can then be repeated n times to extend thesequencing primer by n nucleotides, thereby detecting a sequence oflength n.

While SBS has been described in detail, it is to be understood that theflow cells described herein may be utilized with other sequencingprotocol, for genotyping, or in other chemical and/or biologicalapplications.

To further illustrate the present disclosure, examples are given herein.It is to be understood that these examples are provided for illustrativepurposes and are not to be construed as limiting the scope of thepresent disclosure.

EXAMPLES Example 1

PAZAM with pre-grafted P7 and P5U primers was deposited on a flow cellsubstrate having pillar-like features that were about 400 nm tall andabout 350 nm in diameter. A scanning electron micrograph (SEM) image ofa portion of the deposited pre-grafted PAZAM layer is shown in FIG. 39A.A lift-off resist was deposited on this first grafted PAZAM layer toform an example of the protection layer described herein. A SEM image ofa portion of the deposited protection layer is shown in FIG. 39B.Etching was then performed to remove the protection layer and the firstgrafted PAZAM layer from the tops of the pillar-like features. A SEMimage of a portion of the substrate after etching is shown in FIG. 39C.PAZAM was deposited on the tops of the pillar-like features and on theremaining protection layer to form a second PAZAM layer. The secondPAZAM layer was grafted with P5 and P7U primers.

A lift-off process was used to remove the protection layer and any ofthe second grafted PAZAM layer from the first grafted PAZAM layer.

Library fragments from the same genome (from the Human genome) wereintroduced to the flow cell. The library fragments included a portionthat was complementary to the P5 of P7 primer sequences, along withindex sequences, and read 1 and read 2 sequences. The library fragmentswere seeded and clustering was performed using bridge amplification.Simultaneous paired-end sequencing was then performed.

At the border area between the pillar-like features and neighboringregions, it was found that about 34% of all the reads were paired.

The reads were extracted from a single location and were aligned to thegenome. FIG. 40A illustrates two R1 reads (R1 and R1′) that are imputedto be simultaneous paired-end reads.

The flow cell was also processed through a sequential paired-endsynthesis (forward strands sequenced and removed followed by reversestrand sequencing). FIG. 40B illustrates the results from FIG. 40A withthe second read (R2) from the sequential paired end synthesis flipped toshow that it indeed matches the simultaneous paired-end read pair. Theclipped portion in R2 may be due to quality drop, whereas the R1 read isa higher quality, unclipped read. These results demonstrate that themethods disclosed herein can produce equivalent information to thestandard paired-end sequencing (FIG. 40B) using simultaneously generatedpairs (FIG. 40A).

Still further, the insert size distributions of the library fragmentswere roughly equivalent for data based on simultaneous paired-endsequencing and standard (sequential) paired end sequencing. Theseresults further demonstrate that the methods disclosed herein, usingsimultaneously generated pairs, can produce equivalent information tothe standard paired-end sequencing.

Example 2

PAZAM was deposited on a planar flow cell substrate and grafted with P7and P5U primers. A protection layer (Shipley 1813 photoresist) wasdeposited on top of this first grafted PAZAM layer. UV light was usedthrough a photomask to expose defined portions of the protection layer,which were then developed away in solvent, leaving behind 50 μm circularpads of the protective layer. Air plasma was used to etch away theexposed portions of the first grafted PAZAM layer in the interstitialregions, while the portions under the protective pads remained intact. Asecond layer of PAZAM was then deposited on the interstitial regions andon the remaining protection layer. This second deposited PAZAM was thengrafted with P5 and P7U primers.

A lift-off process was used to remove the protection layer and any ofthe second grafted PAZAM layer from the first grafted PAZAM layer.

Library fragments from the same genome (from the Human genome) wereintroduced to the flow cell. The library fragments included a portionthat was complementary to the P5 of P7 primer sequences, along withindex sequences, and read 1 and read 2 sequences. The library fragmentswere seeded and clustering was performed using bridge amplification.Simultaneous paired-end sequencing was then performed.

At the border area between the first grafted and second grafted PAZAMs,it was found that about 2.2% of all the reads were paired. This meantthat two reads were within 2 μm of each other and were within 2 kb ofeach other in the genome.

Additional Notes

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

Reference throughout the specification to “one example”, “anotherexample”, “an example”, and so forth, means that a particular element(e.g., feature, structure, and/or characteristic) described inconnection with the example is included in at least one exampledescribed herein, and may or may not be present in other examples. Inaddition, it is to be understood that the described elements for anyexample may be combined in any suitable manner in the various examplesunless the context clearly dictates otherwise.

It is to be understood that the ranges provided herein include thestated range and any value or sub-range within the stated range, as ifsuch values or sub-ranges were explicitly recited. For example, a rangeof about 400 nm to about 1 μm (1000 nm), should be interpreted toinclude not only the explicitly recited limits of about 400 nm to about1 μm, but also to include individual values, such as about 708 nm, about945.5 nm, etc., and sub-ranges, such as from about 425 nm to about 825nm, from about 550 nm to about 940 nm, etc. Furthermore, when “about”and/or “substantially” are/is utilized to describe a value, they aremeant to encompass minor variations (up to +/−10%) from the statedvalue.

While several examples have been described in detail, it is to beunderstood that the disclosed examples may be modified. Therefore, theforegoing description is to be considered non-limiting.

1. A flow cell, comprising: a substrate; a first primer set attached toa first region on the substrate, the first primer set including anun-cleavable first primer and a cleavable second primer; and a secondprimer set attached to a second region on the substrate, the secondprimer set including a cleavable first primer and an un-cleavable secondprimer.
 2. The flow cell as defined in claim 1, wherein: the firstregion includes a material having a first functional group; and thesecond region includes a material having a second functional group thatis different than the first functional group.
 3. The flow cell asdefined in claim 1, further comprising a gap separating the first primerset from the second primer set.
 4. The flow cell as defined in claim 1,wherein: the substrate includes depressions separated by interstitialregions; and each of the depressions includes: the first region locatedat a first portion; and the second region located at a second portion.5. The flow cell as defined in claim 4, further comprising a gapseparating the first region from the second region.
 6. The flow cell asdefined in claim 4, wherein the first region and the second regionpartially overlap.
 7. The flow cell as defined in claim 4, wherein thefirst and second portions have different depths.
 8. The flow cell asdefined in claim 4, wherein the first and second regions are differentblocks of a block co-polymer.
 9. The flow cell as defined in claim 1,wherein: the substrate includes depressions separated by interstitialregions; each of the depressions includes the first region; and thesecond region is located on at least some of the interstitial regions.10. The flow cell as defined in claim 1, wherein: the first regionincludes a first polymer and the first primer set is grafted to thefirst polymer; and the second region includes a second polymer and thesecond primer set is grafted to the second polymer.
 11. The flow cell asdefined in claim 10, wherein the flow cell further comprises aprotective coating on the first primer set and on the first polymer. 12.The flow cell as defined in claim 10, wherein: the first polymer is afirst layer on the substrate; the second polymer is a second layer onthe first layer; the flow cell further comprises: a passivation resin onthe second layer; and features defined in the passivation resin, thesecond polymer and the first polymer; and each of the first and secondprimer sets is exposed at each of the features.
 13. The flow cell asdefined in claim 1, wherein: the substrate includes depressionsseparated by interstitial regions; each of the depressions includes: afirst portion where the first region is located; and a second portion;and the flow cell further comprises a bead located in the secondportion, wherein the second region is at a surface of the bead.
 14. Theflow cell as defined in claim 1, wherein the cleavable first primerincludes a first cleavage site, the cleavable second primer includes asecond cleavage site, and the first and second cleavage sites are of anidentical type.
 15. The flow cell as defined in claim 14, wherein: eachof the un-cleavable first primer, the cleavable second primer, thecleavable first primer, and the cleavable second primer includes arespective linker; the first cleavage site of the first cleavable primeris located along its linker; and the second cleavage site of the secondcleavable primer is located along its linker.
 16. The flow cell asdefined in claim 1, wherein the cleavable first primer includes a firstcleavage site, the cleavable second primer includes a second cleavagesite, and the first and second cleavage sites are of a different type.17. The flow cell as defined in claim 16, wherein: each of theun-cleavable first primer, the cleavable second primer, the cleavablefirst primer, and the cleavable second primer includes a respectivelinker; the first cleavage site of the first cleavable primer is locatedalong its linker; and the second cleavage site of the second cleavableprimer is located along its linker.
 18. The flow cell as defined inclaim 1, wherein: the first primer set is attached to a first supportstructure; the first region is a first capture site that is attached tothe first support structure; the second primer set is attached to asecond support structure that is different than the first supportstructure; and the second region is a second capture site that isattached to the second support structure.
 19. A flow cell, comprising: afirst substrate; a first primer set attached to the first substrate, thefirst primer set including an un-cleavable first primer and a cleavablesecond primer; a second substrate opposed to the first substrate; and asecond primer set attached to the second substrate, the second primerset including a cleavable first primer and an un-cleavable secondprimer.
 20. (canceled)
 21. A kit, comprising: a flow cell including: asubstrate including depressions separated by interstitial regions; afirst polymer layer in each of the depressions, wherein some functionalgroups of the first polymer layer are capped; a first primer setattached to other functional groups of first polymer layer in each ofthe depressions; and a second polymer layer on the interstitial regions;and a priming fluid including: a fluid carrier; and a second primer setthat is different from the first primer set; wherein: the first primerset includes an un-cleavable first primer and a cleavable second primer;and the second primer set includes a cleavable first primer and anun-cleavable second primer.
 22. A method, comprising: introducing atemplate fluid to a flow cell including: a substrate includingdepressions separated by interstitial regions; a first polymer layer ineach of the depressions, wherein exposed functional groups of the firstpolymer layer are capped; a first primer set attached to the firstpolymer layer in each of the depressions, the first primer set includinga cleavable first primer and an un-cleavable second primer; and a secondpolymer layer on the interstitial regions; whereby a template from thetemplate fluid is amplified to form a cluster in at least some of thedepressions; introducing a priming fluid, including an un-cleavablefirst primer and a cleavable second primer, to the flow cell, wherebythe un-cleavable first primer and the cleavable second primer graft tothe second polymer layer; and initiating bridge amplification from thecluster to the un-cleavable first primer and a cleavable second primer,thereby forming a second cluster on at least some of the interstitialregions.
 23. A method, comprising: introducing a template fluid to aflow cell including: a substrate including depressions separated byinterstitial regions; a first polymer layer in each of the depressions;a first primer set attached to the first polymer layer, the first primerset including a cleavable first primer and an un-cleavable secondprimer; an optional protective coating layer on the first polymer layerand on the first primer set; a second polymer layer on the interstitialregions; and a second primer set attached to the second polymer layer,the second primer set including an un-cleavable first primer and acleavable second primer; whereby a template from the template fluid isamplified to form a cluster in at least some of the depressions and onat least some of the interstitial regions; and cleaving the cleavablefirst primer and the cleavable second primer. 24.-88. (canceled)